anti icam 1  (Millipore)


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    Structured Review

    Millipore anti icam 1
    In vitro blockade of the <t>ICAM-1/LFA-1</t> pair abolishes T cell clustering and speeds their transmigration across lymphatic monolayers. (A) Representative microphotographs of CD45.1+ CD8+ T cell clusters after in vitro activation for 24 or 48 h in the presence of control isotype or anti-ICAM-1 or anti-LFA-1 antibodies. (B) Quantification of T cell cluster formation in A calculated as the percentage of total area that is occupied by T-cell clusters. Pooled data from at least two independent experiments are shown. (C) Representative microphotographs showing destruction of already-formed T cell clusters of CD45.1+ CD8+ activated T lymphocytes being subsequently treated for 24 h with isotype control, anti-ICAM-1 or anti-LFA-1 antibodies. (D) Quantification of T cell clusters was performed as in B . (E) Transmigration of activated CD45.1+ CD8+ T cells for 16 h across monolayers of IMLEC through serum-containing medium. The assays were performed in the presence of anti-ICAM-1 or anti-LFA-1 antibodies or control IgG. Data from five independent experiments are shown. (F) Percentages of CD8+ T-lymphocytes remaining in the upper well of Boyden chambers after transmigration assays as in E. Percentages of total area occupied by T-cell clusters was calculated using a manual region of interest (ROI) based on CD8+ signal. At least nine pictures where analyzed in each case. (G) Representative confocal images of OT1 CD8+ lymphocytes (red) that remained in the upper well of transwell Boyden chambers after transmigration experiments as in E (* p
    Anti Icam 1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "ICAM-1-LFA-1 Dependent CD8+ T-Lymphocyte Aggregation in Tumor Tissue Prevents Recirculation to Draining Lymph Nodes"

    Article Title: ICAM-1-LFA-1 Dependent CD8+ T-Lymphocyte Aggregation in Tumor Tissue Prevents Recirculation to Draining Lymph Nodes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02084

    In vitro blockade of the ICAM-1/LFA-1 pair abolishes T cell clustering and speeds their transmigration across lymphatic monolayers. (A) Representative microphotographs of CD45.1+ CD8+ T cell clusters after in vitro activation for 24 or 48 h in the presence of control isotype or anti-ICAM-1 or anti-LFA-1 antibodies. (B) Quantification of T cell cluster formation in A calculated as the percentage of total area that is occupied by T-cell clusters. Pooled data from at least two independent experiments are shown. (C) Representative microphotographs showing destruction of already-formed T cell clusters of CD45.1+ CD8+ activated T lymphocytes being subsequently treated for 24 h with isotype control, anti-ICAM-1 or anti-LFA-1 antibodies. (D) Quantification of T cell clusters was performed as in B . (E) Transmigration of activated CD45.1+ CD8+ T cells for 16 h across monolayers of IMLEC through serum-containing medium. The assays were performed in the presence of anti-ICAM-1 or anti-LFA-1 antibodies or control IgG. Data from five independent experiments are shown. (F) Percentages of CD8+ T-lymphocytes remaining in the upper well of Boyden chambers after transmigration assays as in E. Percentages of total area occupied by T-cell clusters was calculated using a manual region of interest (ROI) based on CD8+ signal. At least nine pictures where analyzed in each case. (G) Representative confocal images of OT1 CD8+ lymphocytes (red) that remained in the upper well of transwell Boyden chambers after transmigration experiments as in E (* p
    Figure Legend Snippet: In vitro blockade of the ICAM-1/LFA-1 pair abolishes T cell clustering and speeds their transmigration across lymphatic monolayers. (A) Representative microphotographs of CD45.1+ CD8+ T cell clusters after in vitro activation for 24 or 48 h in the presence of control isotype or anti-ICAM-1 or anti-LFA-1 antibodies. (B) Quantification of T cell cluster formation in A calculated as the percentage of total area that is occupied by T-cell clusters. Pooled data from at least two independent experiments are shown. (C) Representative microphotographs showing destruction of already-formed T cell clusters of CD45.1+ CD8+ activated T lymphocytes being subsequently treated for 24 h with isotype control, anti-ICAM-1 or anti-LFA-1 antibodies. (D) Quantification of T cell clusters was performed as in B . (E) Transmigration of activated CD45.1+ CD8+ T cells for 16 h across monolayers of IMLEC through serum-containing medium. The assays were performed in the presence of anti-ICAM-1 or anti-LFA-1 antibodies or control IgG. Data from five independent experiments are shown. (F) Percentages of CD8+ T-lymphocytes remaining in the upper well of Boyden chambers after transmigration assays as in E. Percentages of total area occupied by T-cell clusters was calculated using a manual region of interest (ROI) based on CD8+ signal. At least nine pictures where analyzed in each case. (G) Representative confocal images of OT1 CD8+ lymphocytes (red) that remained in the upper well of transwell Boyden chambers after transmigration experiments as in E (* p

    Techniques Used: In Vitro, Transmigration Assay, Activation Assay

    In vivo activated T-cells experience increased migration from tumors to the LNs after ICAM-1 blockade. (A) Treatment schedule. (B) Percentages of intravenously injected CD45.1+ CD8+ T cells in the draining LNs 48 h after treatment with isotype ( n = 9) or anti-ICAM-1 ( n = 6) antibodies of mice bearing B16-VEGFC-OVA tumors. Cells were detected by flow cytometry. (C) Percentages of intravenously injected CD45.1+ CD8+ T cells that remained in B16-VEGFC-OVA tumors 48 h after treatment with isotype ( n = 9) or anti-ICAM-1 ( n = 6) antibodies and detected by flow cytometry. (D) Mean fluorescence intensity of CCR7 expression in CD45.1+ CD8+ T cells remaining in B16-VEGFC-OVA tumors in mice treated as in C. (* p
    Figure Legend Snippet: In vivo activated T-cells experience increased migration from tumors to the LNs after ICAM-1 blockade. (A) Treatment schedule. (B) Percentages of intravenously injected CD45.1+ CD8+ T cells in the draining LNs 48 h after treatment with isotype ( n = 9) or anti-ICAM-1 ( n = 6) antibodies of mice bearing B16-VEGFC-OVA tumors. Cells were detected by flow cytometry. (C) Percentages of intravenously injected CD45.1+ CD8+ T cells that remained in B16-VEGFC-OVA tumors 48 h after treatment with isotype ( n = 9) or anti-ICAM-1 ( n = 6) antibodies and detected by flow cytometry. (D) Mean fluorescence intensity of CCR7 expression in CD45.1+ CD8+ T cells remaining in B16-VEGFC-OVA tumors in mice treated as in C. (* p

    Techniques Used: In Vivo, Migration, Injection, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Expressing

    Integrin ligand blockade leads to increments in chemokine-dependent migration. (A) Surface expression of the chemokine receptor CCR7 on in vitro activated OT-1 CD8+ T cells in the presence or in the absence of anti-ICAM-1 antibodies. Flow-cytometry data was collected from three or more independent experiments. (B) Chemokine-directed migration of activated CD45.1+ CD8+ T lymphocytes in 3D collagen gels after being treated with anti-ICAM-1 antibodies or control IgG for 48 h. Cell movement toward CCL21 gradients was recorded by in vivo confocal microscopy and cell straightness was analyzed using IMARIS software. Each dot represents a single tracked cell. At least 80 cells were tracked in each case. (C) Percentages of intratumorally-injected CD45.1+ CD8+ T cells that reached the draining LNs 48 h after treatment with isotype antibody ( n = 35), isotype + PTx ( n = 6), anti-ICAM-1 antibody ( n = 39), or anti-ICAM-1 + PTx ( n = 10). (D) Mean fluorescence intensity of CCR7 expression in LN CD45.1+ CD8+ T cells 48 h after treatment with isotype ( n = 26) or anti-ICAM-1 ( n = 33) antibodies. Cells were detected by flow cytometry (** p
    Figure Legend Snippet: Integrin ligand blockade leads to increments in chemokine-dependent migration. (A) Surface expression of the chemokine receptor CCR7 on in vitro activated OT-1 CD8+ T cells in the presence or in the absence of anti-ICAM-1 antibodies. Flow-cytometry data was collected from three or more independent experiments. (B) Chemokine-directed migration of activated CD45.1+ CD8+ T lymphocytes in 3D collagen gels after being treated with anti-ICAM-1 antibodies or control IgG for 48 h. Cell movement toward CCL21 gradients was recorded by in vivo confocal microscopy and cell straightness was analyzed using IMARIS software. Each dot represents a single tracked cell. At least 80 cells were tracked in each case. (C) Percentages of intratumorally-injected CD45.1+ CD8+ T cells that reached the draining LNs 48 h after treatment with isotype antibody ( n = 35), isotype + PTx ( n = 6), anti-ICAM-1 antibody ( n = 39), or anti-ICAM-1 + PTx ( n = 10). (D) Mean fluorescence intensity of CCR7 expression in LN CD45.1+ CD8+ T cells 48 h after treatment with isotype ( n = 26) or anti-ICAM-1 ( n = 33) antibodies. Cells were detected by flow cytometry (** p

    Techniques Used: Migration, Expressing, In Vitro, Flow Cytometry, Cytometry, In Vivo, Confocal Microscopy, Software, Injection, Fluorescence

    Intratumoral CD8+ T-cells form diffuse cell-clusters that are reduced upon anti-ICAM-1 treatment. (A) Representative confocal images of B16-VEGFC tumors from isotype or anti-ICAM-1 treated mice showing CD45.1+ T cells (orange), CD8+ T cells (red), and intratumoral LVs (green). Images at the right end show magnifications of T cell clusters. (B) Percentages of area occupied by CD45.1+ CD8+ T-lymphocyte clusters in B16-VEGFC tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 13 pictures where analyzed in each case. (C) Representative confocal images of B16-VEGFC tumors from isotype or anti-LFA-1 treated mice showing CD45.1+ T cells (orange) and CD8+ T cells (red). (D) Percentages of area occupied by CD45.1+ CD8+ T-lymphocyte clusters in B16-VEGFC tumors 48 h after isotype or anti-LFA-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 19 pictures where analyzed in each case. (E) Representative confocal images of Her2/Neu tumors in isotype or anti-ICAM-1 treated mice showing CD8+ T cells (red). (F) Percentages of area occupied by CD8+ T-lymphocyte clusters in Her2/Neu tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 26 pictures where analyzed in each case. (G) Representative microphotograph showing CD8+ T cell clusters as detected by immunohistochemistry on samples obtained from a series of 10 human melanoma patients. Magnifications show two instances of T cell clusters (* p
    Figure Legend Snippet: Intratumoral CD8+ T-cells form diffuse cell-clusters that are reduced upon anti-ICAM-1 treatment. (A) Representative confocal images of B16-VEGFC tumors from isotype or anti-ICAM-1 treated mice showing CD45.1+ T cells (orange), CD8+ T cells (red), and intratumoral LVs (green). Images at the right end show magnifications of T cell clusters. (B) Percentages of area occupied by CD45.1+ CD8+ T-lymphocyte clusters in B16-VEGFC tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 13 pictures where analyzed in each case. (C) Representative confocal images of B16-VEGFC tumors from isotype or anti-LFA-1 treated mice showing CD45.1+ T cells (orange) and CD8+ T cells (red). (D) Percentages of area occupied by CD45.1+ CD8+ T-lymphocyte clusters in B16-VEGFC tumors 48 h after isotype or anti-LFA-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 19 pictures where analyzed in each case. (E) Representative confocal images of Her2/Neu tumors in isotype or anti-ICAM-1 treated mice showing CD8+ T cells (red). (F) Percentages of area occupied by CD8+ T-lymphocyte clusters in Her2/Neu tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 26 pictures where analyzed in each case. (G) Representative microphotograph showing CD8+ T cell clusters as detected by immunohistochemistry on samples obtained from a series of 10 human melanoma patients. Magnifications show two instances of T cell clusters (* p

    Techniques Used: Mouse Assay, Immunohistochemistry

    ICAM-1-LFA-1 dependent CD8+ T-lymphocyte aggregation in tumor tissue prevents recirculation to draining lymph nodes. (A) Intra-tumoral CD8+ T lymphocytes form clusters that are retained in the tumor microenvironment by homotypic ICAM-1-LFA-1 interactions. (B) ICAM-1 blockade results in destruction of T-cell clusters and increased T cell sensitivity to CCL21 chemokine-guided migration toward the lymphatic vessels, thus facilitating their transit to the draining lymph nodes.
    Figure Legend Snippet: ICAM-1-LFA-1 dependent CD8+ T-lymphocyte aggregation in tumor tissue prevents recirculation to draining lymph nodes. (A) Intra-tumoral CD8+ T lymphocytes form clusters that are retained in the tumor microenvironment by homotypic ICAM-1-LFA-1 interactions. (B) ICAM-1 blockade results in destruction of T-cell clusters and increased T cell sensitivity to CCL21 chemokine-guided migration toward the lymphatic vessels, thus facilitating their transit to the draining lymph nodes.

    Techniques Used: Migration

    Systemic ICAM-1 blockade increases the egress of intratumorally injected CD8+ T-cells to draining LNs. (A) Percentages of total CD45.1+ CD8+ T cells injected in B16-VEGFC tumors that reached the draining LNs 48 h after isotype ( n = 29), anti-ICAM-1 ( n = 32), or anti-LFA-1 ( n = 12) treatment and detected by flow cytometry. (B) Representative confocal images of B16-VEGFC tumors in mice treated with isotype, anti-ICAM-1 or anti-LFA-1 antibodies showing CD45.1+ T cells (red) and intratumoral LVs (green). (C) Percentages of CD45.1+ CD8+ T-lymphocytes remaining in B16-VEGFC tumors 48 h after isotype, anti-ICAM-1 or anti-LFA-1 antibody treatment. Percentage of positive area was calculated using a manual region of interest (ROI) based on CD45.1 signal. At least 12 pictures from 3 different tumors where analyzed in each case. (D) Percentages of total CD8+ T cells injected in Her2/Neu tumors that reached the draining LNs after isotype ( n = 6) or anti-ICAM-1 ( n = 8) treatment and detected by flow cytometry. (E) Percentages of total CD8+ T cells injected in Her2/Neu tumors that remained in tumors 48 h after isotype ( n = 6) or anti-ICAM-1 ( n = 8) treatment and detected by flow cytometry. (F) Percentages of CD8+ T-lymphocytes remaining in Her2/Neu tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of positive area was calculated using a manual region of interest (ROI) based on CD8 signal. At least 23 pictures from 3 different tumors where analyzed in each case (* p
    Figure Legend Snippet: Systemic ICAM-1 blockade increases the egress of intratumorally injected CD8+ T-cells to draining LNs. (A) Percentages of total CD45.1+ CD8+ T cells injected in B16-VEGFC tumors that reached the draining LNs 48 h after isotype ( n = 29), anti-ICAM-1 ( n = 32), or anti-LFA-1 ( n = 12) treatment and detected by flow cytometry. (B) Representative confocal images of B16-VEGFC tumors in mice treated with isotype, anti-ICAM-1 or anti-LFA-1 antibodies showing CD45.1+ T cells (red) and intratumoral LVs (green). (C) Percentages of CD45.1+ CD8+ T-lymphocytes remaining in B16-VEGFC tumors 48 h after isotype, anti-ICAM-1 or anti-LFA-1 antibody treatment. Percentage of positive area was calculated using a manual region of interest (ROI) based on CD45.1 signal. At least 12 pictures from 3 different tumors where analyzed in each case. (D) Percentages of total CD8+ T cells injected in Her2/Neu tumors that reached the draining LNs after isotype ( n = 6) or anti-ICAM-1 ( n = 8) treatment and detected by flow cytometry. (E) Percentages of total CD8+ T cells injected in Her2/Neu tumors that remained in tumors 48 h after isotype ( n = 6) or anti-ICAM-1 ( n = 8) treatment and detected by flow cytometry. (F) Percentages of CD8+ T-lymphocytes remaining in Her2/Neu tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of positive area was calculated using a manual region of interest (ROI) based on CD8 signal. At least 23 pictures from 3 different tumors where analyzed in each case (* p

    Techniques Used: Injection, Flow Cytometry, Cytometry, Mouse Assay

    2) Product Images from "WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts"

    Article Title: WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts

    Journal: Journal of Leukocyte Biology

    doi: 10.1189/jlb.2A0215-050RR

    The WASp-dependent DC actin cytoskeleton contributes to correct organization of adhesion molecules and formation of an extensive cell:cell contact. (A) WT, WASKO, and Y293F DCs (yellow/orange), pulsed with OVA, were cocultured with OT-II T cells (blue/green). After 1 h, conjugates were fixed, processed, and imaged using Gatan 3View. Isosurface reconstructions were created in Amira. (Lower) Conjugates with T cell removed to visualize the contact interface. (B) Quantification of DC:T cell contact surface area as a percentage of T cell surface. A minimum of 10 conjugates was analyzed per group. Unpaired t test was used to test significance among DC types; *** P (WT + WASKO) = 0.0005, P (WASKO + Y293F) = 0.0002; ns P (WT + Y293F) = 0.3153. (C and D) DCs expressing ICAM-1-GFP (green) were cocultured with T cells (red; anti-TCR immunostain) for 45 min and fixed. Images represent a slice cutting through the synapse. Polarization ratios of ICAM-1 on the DC side were calculated by measuring fluorescence intensity at the synapse normalized to whole cell. Original scale bars, 5 μm. Unpaired t test was used to test significance among DC types; ** P (WT + WASKO) = 0.0026; ns, P (WT + Y293F) = 0.1875, P (WASKO + Y293F) = 0.0610. DIC, Differential interference contrast. (E) Total surface ICAM-1 was measured by flow cytometry in immature and LPS-matured BMDCs, gated on CD11c-positive cells. Means and sd are shown from 3 independent experiments. MFI, Mean fluorescence intensity. (F) Total polymerized actin was measured using phalloidin in permeabilized, immature and LPS-matured BMDCs. Staining was performed in mixed population samples using CFSE labeling. Bars represent means and sd from 3 experiments. *** P (WT + WASKO) = 0.0010, * P (WT + Y293F) = 0.0472, * P (WASKO + Y293F) = 0.0407.
    Figure Legend Snippet: The WASp-dependent DC actin cytoskeleton contributes to correct organization of adhesion molecules and formation of an extensive cell:cell contact. (A) WT, WASKO, and Y293F DCs (yellow/orange), pulsed with OVA, were cocultured with OT-II T cells (blue/green). After 1 h, conjugates were fixed, processed, and imaged using Gatan 3View. Isosurface reconstructions were created in Amira. (Lower) Conjugates with T cell removed to visualize the contact interface. (B) Quantification of DC:T cell contact surface area as a percentage of T cell surface. A minimum of 10 conjugates was analyzed per group. Unpaired t test was used to test significance among DC types; *** P (WT + WASKO) = 0.0005, P (WASKO + Y293F) = 0.0002; ns P (WT + Y293F) = 0.3153. (C and D) DCs expressing ICAM-1-GFP (green) were cocultured with T cells (red; anti-TCR immunostain) for 45 min and fixed. Images represent a slice cutting through the synapse. Polarization ratios of ICAM-1 on the DC side were calculated by measuring fluorescence intensity at the synapse normalized to whole cell. Original scale bars, 5 μm. Unpaired t test was used to test significance among DC types; ** P (WT + WASKO) = 0.0026; ns, P (WT + Y293F) = 0.1875, P (WASKO + Y293F) = 0.0610. DIC, Differential interference contrast. (E) Total surface ICAM-1 was measured by flow cytometry in immature and LPS-matured BMDCs, gated on CD11c-positive cells. Means and sd are shown from 3 independent experiments. MFI, Mean fluorescence intensity. (F) Total polymerized actin was measured using phalloidin in permeabilized, immature and LPS-matured BMDCs. Staining was performed in mixed population samples using CFSE labeling. Bars represent means and sd from 3 experiments. *** P (WT + WASKO) = 0.0010, * P (WT + Y293F) = 0.0472, * P (WASKO + Y293F) = 0.0407.

    Techniques Used: Expressing, Fluorescence, Flow Cytometry, Cytometry, Staining, Labeling

    The development of a novel imaging system of the DC synapse. (A) Three different lipid bilayer compositions were designed to mimic a DC:T cell synapse. A biotinylated α-MHC II-Cy5-conjugated antibody was incorporated to replace TCR interaction ( Fig. 2 ). Alternatively, both α-MHC II-Cy5 and α-ICAM-1 antibodies were added to replicate adhesion forces (A and E). α-ICAM-1 alone was used in D. (B) ICAM-1-GFP-expressing DCs interacting with an α-MHC II bilayer were fixed after 20 min and imaged. Original scale bars, 5 μm. Fluorescence intensity of ICAM-1-GFP and MHC II-Cy5 is plotted along the cell diameter, showing differential distribution in WT and WASKO DC. (C) Number of cells exhibiting radial symmetry, as a percentage of cells interacting with the α-MHC II bilayer. A minimum of 30 cells per strain was analyzed. Cells with radial symmetry were defined as having at least 3 different diameter cross-sections showing plots similar to WT DC in B. * P = 0.0232; ** P = 0.0092; ns, P = 0.0572. (D) WT, WASKO, and Y293F DCs interacting with the α-MHC II-Cy5 (red) bilayer were fixed and stained with phalloidin (blue). Original scale bars, 5 μm. (E) Three parameters were measured by use of ImageJ “Measure” and “Analyze particles” functions in cells interacting with an α-MHC II-Cy5 bilayer: average actin intensity across the contact; MHC II area as a percentage of the total contact area (actin); and number of peripheral microclusters (MC) per cell (size
    Figure Legend Snippet: The development of a novel imaging system of the DC synapse. (A) Three different lipid bilayer compositions were designed to mimic a DC:T cell synapse. A biotinylated α-MHC II-Cy5-conjugated antibody was incorporated to replace TCR interaction ( Fig. 2 ). Alternatively, both α-MHC II-Cy5 and α-ICAM-1 antibodies were added to replicate adhesion forces (A and E). α-ICAM-1 alone was used in D. (B) ICAM-1-GFP-expressing DCs interacting with an α-MHC II bilayer were fixed after 20 min and imaged. Original scale bars, 5 μm. Fluorescence intensity of ICAM-1-GFP and MHC II-Cy5 is plotted along the cell diameter, showing differential distribution in WT and WASKO DC. (C) Number of cells exhibiting radial symmetry, as a percentage of cells interacting with the α-MHC II bilayer. A minimum of 30 cells per strain was analyzed. Cells with radial symmetry were defined as having at least 3 different diameter cross-sections showing plots similar to WT DC in B. * P = 0.0232; ** P = 0.0092; ns, P = 0.0572. (D) WT, WASKO, and Y293F DCs interacting with the α-MHC II-Cy5 (red) bilayer were fixed and stained with phalloidin (blue). Original scale bars, 5 μm. (E) Three parameters were measured by use of ImageJ “Measure” and “Analyze particles” functions in cells interacting with an α-MHC II-Cy5 bilayer: average actin intensity across the contact; MHC II area as a percentage of the total contact area (actin); and number of peripheral microclusters (MC) per cell (size

    Techniques Used: Imaging, Expressing, Fluorescence, Staining

    Novel actin organization at the DC synapse. (A) WT, WASKO, and Y293F DCs interacting with an α-MHC II-Cy5 (red) and α-ICAM-1 bilayer were fixed and stained with phalloidin (blue). Original scale bars, 5 μm. (B) The polymerized actin was stained with phalloidin, and fluorescent intensity at the contact site as well as total actin area was quantified at the 4 time points. A minimum of 100 cells was analyzed in 4 experiments; means and sem are plotted. For actin intensity: *5 min: P (WT + WASKO) = 0.0113, P (WT + Y293F) = 0.0428; ***15 min: P (WT + WASKO) = 0.0005, P (WASKO + Y293F) = 0.0256; ***30 min: P (WT + WASKO) = 0.0007; ***60 min: P (WT + WASKO) = 0.0009. For total actin area: *15 min: P (WT + WASKO) = 0.0346. (C) Percentage of WT, WASKO, and Y293F DCs forming podosomes on an α-MHC II and α-ICAM-1 bilayer. A minimum of 400 cells was analyzed at each time point. “Actin clusters” are irregular, high-intensity actin structures, similar to those in WASKO cells at 30 and 60 min (A). (D) WT DC contacting an α-ICAM-1-only bilayer. Position of cells is depicted by DAPI staining (white). Phalloidin staining (blue) shows podosome rosettes. (E) The proportion of WT cells forming rings of podosomes in the 3 bilayer conditions is quantified. Means and sem from 3 experiments are shown; a minimum of 300 cells was analyzed. * P = 0.0241, ** P = 0.0073 (F) WT DC contacting an α-MHC II-Cy5 and α-ICAM-1 bilayer, showing actin-rich podosomes (blue) and immunofluorescent staining (yellow): capping protein (F-actin capping protein, α subunit; upper), vinculin (lower). Colocalization of F-actin capping protein and actin produces a white overlay; 36 WT DCs were analyzed to calculate colocalization. Pearson correlation coefficient = 0.442 ± 0.14; Mander’s overlap coefficient = 0.777 ± 0.04. Original scale bars, 5 μm. A 3× zoom is shown to the right. (G) DCs were seeded on 2 different bilayers and on fibronectin (50 μg/ml) and fixed at set intervals. Diameter of the podosome actin cores was measured in ImageJ; > 100 podosomes were measured for each condition. Synapse podosomes did not change significantly over time and showed a similar size to those formed on the ventral side of cells adhering to fibronectin.
    Figure Legend Snippet: Novel actin organization at the DC synapse. (A) WT, WASKO, and Y293F DCs interacting with an α-MHC II-Cy5 (red) and α-ICAM-1 bilayer were fixed and stained with phalloidin (blue). Original scale bars, 5 μm. (B) The polymerized actin was stained with phalloidin, and fluorescent intensity at the contact site as well as total actin area was quantified at the 4 time points. A minimum of 100 cells was analyzed in 4 experiments; means and sem are plotted. For actin intensity: *5 min: P (WT + WASKO) = 0.0113, P (WT + Y293F) = 0.0428; ***15 min: P (WT + WASKO) = 0.0005, P (WASKO + Y293F) = 0.0256; ***30 min: P (WT + WASKO) = 0.0007; ***60 min: P (WT + WASKO) = 0.0009. For total actin area: *15 min: P (WT + WASKO) = 0.0346. (C) Percentage of WT, WASKO, and Y293F DCs forming podosomes on an α-MHC II and α-ICAM-1 bilayer. A minimum of 400 cells was analyzed at each time point. “Actin clusters” are irregular, high-intensity actin structures, similar to those in WASKO cells at 30 and 60 min (A). (D) WT DC contacting an α-ICAM-1-only bilayer. Position of cells is depicted by DAPI staining (white). Phalloidin staining (blue) shows podosome rosettes. (E) The proportion of WT cells forming rings of podosomes in the 3 bilayer conditions is quantified. Means and sem from 3 experiments are shown; a minimum of 300 cells was analyzed. * P = 0.0241, ** P = 0.0073 (F) WT DC contacting an α-MHC II-Cy5 and α-ICAM-1 bilayer, showing actin-rich podosomes (blue) and immunofluorescent staining (yellow): capping protein (F-actin capping protein, α subunit; upper), vinculin (lower). Colocalization of F-actin capping protein and actin produces a white overlay; 36 WT DCs were analyzed to calculate colocalization. Pearson correlation coefficient = 0.442 ± 0.14; Mander’s overlap coefficient = 0.777 ± 0.04. Original scale bars, 5 μm. A 3× zoom is shown to the right. (G) DCs were seeded on 2 different bilayers and on fibronectin (50 μg/ml) and fixed at set intervals. Diameter of the podosome actin cores was measured in ImageJ; > 100 podosomes were measured for each condition. Synapse podosomes did not change significantly over time and showed a similar size to those formed on the ventral side of cells adhering to fibronectin.

    Techniques Used: Staining

    3) Product Images from "Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac–/ICAM-1-dependent mechanism"

    Article Title: Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac–/ICAM-1-dependent mechanism

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0802167

    Interaction of TNFα-matured DCs with ICAM-1 expressed by LECs decreases expression of CD86 in the absence of antigen
    Figure Legend Snippet: Interaction of TNFα-matured DCs with ICAM-1 expressed by LECs decreases expression of CD86 in the absence of antigen

    Techniques Used: Expressing

    Expression of ICAM-1 on collecting lymphatic vessels and on lymphatic sinuses in mouse lymph node
    Figure Legend Snippet: Expression of ICAM-1 on collecting lymphatic vessels and on lymphatic sinuses in mouse lymph node

    Techniques Used: Expressing

    Expression of ICAM-1 on LECs in vitro
    Figure Legend Snippet: Expression of ICAM-1 on LECs in vitro

    Techniques Used: Expressing, In Vitro

    imDCs bind ICAM-1 via Mac-1
    Figure Legend Snippet: imDCs bind ICAM-1 via Mac-1

    Techniques Used:

    The effect of ICAM-1 deficiency on CD86 expression by DCs migrated into regional lymph nodes
    Figure Legend Snippet: The effect of ICAM-1 deficiency on CD86 expression by DCs migrated into regional lymph nodes

    Techniques Used: Expressing

    4) Product Images from "ICAM-1/LFA-1 interaction contributes to the induction of endothelial cell-cell separation: implication for enhanced leukocyte diapedesis"

    Article Title: ICAM-1/LFA-1 interaction contributes to the induction of endothelial cell-cell separation: implication for enhanced leukocyte diapedesis

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2009.41.5.038

    Effects of ICAM-1 expression on EC height and contractility. (A) The monolayers of HUVECs were incubated with TNF-α for 0 h or 24 h at 37℃. Cells were fixed and stained with ICAM-1 (R6.5; green), ZO-1 (green), PECAM-1 (green) and actin
    Figure Legend Snippet: Effects of ICAM-1 expression on EC height and contractility. (A) The monolayers of HUVECs were incubated with TNF-α for 0 h or 24 h at 37℃. Cells were fixed and stained with ICAM-1 (R6.5; green), ZO-1 (green), PECAM-1 (green) and actin

    Techniques Used: Expressing, Incubation, Staining

    ICAM-1/LFA-1 interaction is essential for leukocyte-induced cell-cell separation between ECs. PBLs (5 × 10 5 cells) stimulated with SDF-α were incubated on monolayers of HUVECs with or without TNF-α stimulation and monitored by
    Figure Legend Snippet: ICAM-1/LFA-1 interaction is essential for leukocyte-induced cell-cell separation between ECs. PBLs (5 × 10 5 cells) stimulated with SDF-α were incubated on monolayers of HUVECs with or without TNF-α stimulation and monitored by

    Techniques Used: Incubation

    5) Product Images from "LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro"

    Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-12-455

    Effect of CD11 and CD18-blocking antibodies on the formation of clumps. A Semi-quantitative PCRs were performed to detect the expression of the ICAM-1 transcript. GAPDH is used as a DNA amount control. B A375, 1205LU and SLM8 cell lines were treated with 2 μg/ml of CD11a or CD18-blocking antibodies as indicated. Melanoma cells were labeled with DiO then fixed and labeled with DAPI prior to their observation under an epifluorescence microscope using a magnification of x10. Data were obtained from 3 independent experiments.
    Figure Legend Snippet: Effect of CD11 and CD18-blocking antibodies on the formation of clumps. A Semi-quantitative PCRs were performed to detect the expression of the ICAM-1 transcript. GAPDH is used as a DNA amount control. B A375, 1205LU and SLM8 cell lines were treated with 2 μg/ml of CD11a or CD18-blocking antibodies as indicated. Melanoma cells were labeled with DiO then fixed and labeled with DAPI prior to their observation under an epifluorescence microscope using a magnification of x10. Data were obtained from 3 independent experiments.

    Techniques Used: Blocking Assay, Expressing, Labeling, Microscopy

    Conditioned mediums from melanoma cell lines enhance transcript expression of ICAM-1 in HUVEC cells. Semi-quantitative PCRs were performed to detect expression of ICAM-1 transcripts. A HUVEC cells were treated either with TNF-α and IFN-γ at 100ng/ml or B with conditioned medium from A375 (H+A375), SLM8 (H+SLM8) and 1205LU (H+1205LU) after 48hrs of cell culture. GAPDH is used as a DNA amount control. Data were obtained from 3 independent experiments.
    Figure Legend Snippet: Conditioned mediums from melanoma cell lines enhance transcript expression of ICAM-1 in HUVEC cells. Semi-quantitative PCRs were performed to detect expression of ICAM-1 transcripts. A HUVEC cells were treated either with TNF-α and IFN-γ at 100ng/ml or B with conditioned medium from A375 (H+A375), SLM8 (H+SLM8) and 1205LU (H+1205LU) after 48hrs of cell culture. GAPDH is used as a DNA amount control. Data were obtained from 3 independent experiments.

    Techniques Used: Expressing, Cell Culture

    Effect of ICAM-1-blocking antibodies on the transendothelial migration of A375, 1205LU and SLM8 cell lines. The experiments were performed as detailed in Figure 1 , except that 2μg/ml of ICAM-1-blocking antibodies were introduced in the upper chamber of the Transwell when indicated. Histograms represent 3 independent experiments. In each experiment each condition was analyzed in duplicate.
    Figure Legend Snippet: Effect of ICAM-1-blocking antibodies on the transendothelial migration of A375, 1205LU and SLM8 cell lines. The experiments were performed as detailed in Figure 1 , except that 2μg/ml of ICAM-1-blocking antibodies were introduced in the upper chamber of the Transwell when indicated. Histograms represent 3 independent experiments. In each experiment each condition was analyzed in duplicate.

    Techniques Used: Blocking Assay, Migration

    6) Product Images from "The Transcription Factor Erg Controls Endothelial Cell Quiescence by Repressing Activity of Nuclear Factor (NF)-?B p65 *"

    Article Title: The Transcription Factor Erg Controls Endothelial Cell Quiescence by Repressing Activity of Nuclear Factor (NF)-?B p65 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.346791

    Erg represses ICAM-1 and binds to the ICAM-1 promoter region 4 in resting EC. A and B, HUVEC were treated with siRNA for Erg, Fli-1, Ets-2, or GABPα for 24 h; relative expression of Erg, Fli-1, Ets-2, and GABPα mRNA ( A ) or ICAM-1 mRNA ( B ) was quantified by RT-PCR and expressed as relative to control siRNA treatment ( Cont ). C, analysis of 1.3 kb of the ICAM-1 promoter identified putative EBS, Erg consensus sites, AP1/ETS sites, and an NF-κB site as indicated. Location of quantitative PCR amplicons covering R1, R2, R3, R4, and R5 are indicated by black lines below. D and E , ChIP was carried out using an anti-Erg or control IgG antibody on sheared chromatin from confluent resting HUVEC ( D ) or HUVEC treated with Erg or control siRNA ( E ). Immunoprecipitated DNA was analyzed by qPCR for primers covering ICAM-1 promoter regions 1–5 ( D ) or region 4 only ( E ) and negative control region. Results are expressed as fold-change compared with IgG normalized to input and negative control region. n = 6 ( B ), n = 5 ( C ), *, p
    Figure Legend Snippet: Erg represses ICAM-1 and binds to the ICAM-1 promoter region 4 in resting EC. A and B, HUVEC were treated with siRNA for Erg, Fli-1, Ets-2, or GABPα for 24 h; relative expression of Erg, Fli-1, Ets-2, and GABPα mRNA ( A ) or ICAM-1 mRNA ( B ) was quantified by RT-PCR and expressed as relative to control siRNA treatment ( Cont ). C, analysis of 1.3 kb of the ICAM-1 promoter identified putative EBS, Erg consensus sites, AP1/ETS sites, and an NF-κB site as indicated. Location of quantitative PCR amplicons covering R1, R2, R3, R4, and R5 are indicated by black lines below. D and E , ChIP was carried out using an anti-Erg or control IgG antibody on sheared chromatin from confluent resting HUVEC ( D ) or HUVEC treated with Erg or control siRNA ( E ). Immunoprecipitated DNA was analyzed by qPCR for primers covering ICAM-1 promoter regions 1–5 ( D ) or region 4 only ( E ) and negative control region. Results are expressed as fold-change compared with IgG normalized to input and negative control region. n = 6 ( B ), n = 5 ( C ), *, p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control

    NF-κB induces ICAM-1 expression after Erg inhibition. A , Erg siRNA or control siRNA-treated HUVEC were transduced with AdIκBαSR or AdLacZ. ICAM-1 mRNA levels were measured by quantitative RT-PCR and results are expressed as fold-change compared with control siRNA AdLacZ-treated. n = 5. B , Erg or control Genebloc-transfected HUVEC were treated with BAY-11-7085 or dimethyl sulfoxide ( DMSO ) control. ICAM-1 protein levels were measured by Western blot using an anti-ICAM-1 antibody and normalized to levels of GAPDH ( n = 3). *, p
    Figure Legend Snippet: NF-κB induces ICAM-1 expression after Erg inhibition. A , Erg siRNA or control siRNA-treated HUVEC were transduced with AdIκBαSR or AdLacZ. ICAM-1 mRNA levels were measured by quantitative RT-PCR and results are expressed as fold-change compared with control siRNA AdLacZ-treated. n = 5. B , Erg or control Genebloc-transfected HUVEC were treated with BAY-11-7085 or dimethyl sulfoxide ( DMSO ) control. ICAM-1 protein levels were measured by Western blot using an anti-ICAM-1 antibody and normalized to levels of GAPDH ( n = 3). *, p

    Techniques Used: Expressing, Inhibition, Transduction, Quantitative RT-PCR, Transfection, Western Blot

    Erg binds to ICAM-1 promoter through EBS −118 and −181, and NF-κB p65 binds to EBS −181. A , and B , biotinylated oligonucleotides containing sequences from the ICAM-1 promoter EBS −118 ( A ) or the EBS −181 ( B ) were incubated with nuclear lysate from resting HUVEC. Antibodies to Erg, p65, or IgG were incubated with nuclear extract-oligonucleotide complexes as indicated. Specificity was measured by addition of saturating amounts of competing oligonucleotide (competitor) or competing oligonucleotide with a mutation in the EBS −118 ( A ) or −181 ( B ) (M-competitor). Shifted protein-oligonucleotide complexes are indicated by an arrow and super-shifted complexes are indicated by arrowhead . Images are a single representation of at least 3 separate experiments. C and D, ChIP was carried out on sheared chromatin from confluent resting HUVEC ± TNF (10 ng/ml for 30 min) ( C ), or HUVEC treated with Erg or control siRNA ( D ) using an anti-NF-κB p65 or control IgG antibody. Immunoprecipitated DNA was analyzed by qPCR for primers covering ICAM-1 promoter regions (R) 3–5 ( C ) or 4 ( D ) and negative control region. Results are expressed as fold-change compared with IgG normalized to input and negative control region. n = 4 ( C ), n = 6 ( D ); *, p
    Figure Legend Snippet: Erg binds to ICAM-1 promoter through EBS −118 and −181, and NF-κB p65 binds to EBS −181. A , and B , biotinylated oligonucleotides containing sequences from the ICAM-1 promoter EBS −118 ( A ) or the EBS −181 ( B ) were incubated with nuclear lysate from resting HUVEC. Antibodies to Erg, p65, or IgG were incubated with nuclear extract-oligonucleotide complexes as indicated. Specificity was measured by addition of saturating amounts of competing oligonucleotide (competitor) or competing oligonucleotide with a mutation in the EBS −118 ( A ) or −181 ( B ) (M-competitor). Shifted protein-oligonucleotide complexes are indicated by an arrow and super-shifted complexes are indicated by arrowhead . Images are a single representation of at least 3 separate experiments. C and D, ChIP was carried out on sheared chromatin from confluent resting HUVEC ± TNF (10 ng/ml for 30 min) ( C ), or HUVEC treated with Erg or control siRNA ( D ) using an anti-NF-κB p65 or control IgG antibody. Immunoprecipitated DNA was analyzed by qPCR for primers covering ICAM-1 promoter regions (R) 3–5 ( C ) or 4 ( D ) and negative control region. Results are expressed as fold-change compared with IgG normalized to input and negative control region. n = 4 ( C ), n = 6 ( D ); *, p

    Techniques Used: Incubation, Mutagenesis, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    Identification of the DNA binding site involved in Erg-mediated repression of ICAM-1. A , schematic diagram of ICAM-1 promoter mutant constructs. pGL4 ICAM-1 1.3 was mutated in either single or double EBS or in the NF-κB binding site. EBS were mutated from GGAA to CCAA. NF-κB was mutated as previously shown ( 15 ). B , ICAM-1 promoter activity of EBS mutants after Erg Genebloc treatment. Results are expressed as luciferase activity relative to control Genebloc-treated cells. C , ICAM-1 promoter activity of EBS mutants after AdErg treatment, expressed as luciferase activity relative to AdLacZ ( n = 3–7). *, p
    Figure Legend Snippet: Identification of the DNA binding site involved in Erg-mediated repression of ICAM-1. A , schematic diagram of ICAM-1 promoter mutant constructs. pGL4 ICAM-1 1.3 was mutated in either single or double EBS or in the NF-κB binding site. EBS were mutated from GGAA to CCAA. NF-κB was mutated as previously shown ( 15 ). B , ICAM-1 promoter activity of EBS mutants after Erg Genebloc treatment. Results are expressed as luciferase activity relative to control Genebloc-treated cells. C , ICAM-1 promoter activity of EBS mutants after AdErg treatment, expressed as luciferase activity relative to AdLacZ ( n = 3–7). *, p

    Techniques Used: Binding Assay, Mutagenesis, Construct, Activity Assay, Luciferase

    7) Product Images from "Measles Virus-Induced Block of Transendothelial Migration of T Lymphocytes and Infection-Mediated Virus Spread across Endothelial Cell Barriers ▿"

    Article Title: Measles Virus-Induced Block of Transendothelial Migration of T Lymphocytes and Infection-Mediated Virus Spread across Endothelial Cell Barriers ▿

    Journal:

    doi: 10.1128/JVI.00775-08

    Formation of transmigratory cups and infection of endothelial cells. (A) Transmigratory cups visualized with antibodies to ICAM-1 and Alexa-594-conjugated secondary antibodies (red) formed around adhering rMV IC323eGFP -infected leukocytes (green). Left,
    Figure Legend Snippet: Formation of transmigratory cups and infection of endothelial cells. (A) Transmigratory cups visualized with antibodies to ICAM-1 and Alexa-594-conjugated secondary antibodies (red) formed around adhering rMV IC323eGFP -infected leukocytes (green). Left,

    Techniques Used: Infection

    8) Product Images from "ICAM-1-LFA-1 Dependent CD8+ T-Lymphocyte Aggregation in Tumor Tissue Prevents Recirculation to Draining Lymph Nodes"

    Article Title: ICAM-1-LFA-1 Dependent CD8+ T-Lymphocyte Aggregation in Tumor Tissue Prevents Recirculation to Draining Lymph Nodes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02084

    In vitro blockade of the ICAM-1/LFA-1 pair abolishes T cell clustering and speeds their transmigration across lymphatic monolayers. (A) Representative microphotographs of CD45.1+ CD8+ T cell clusters after in vitro activation for 24 or 48 h in the presence of control isotype or anti-ICAM-1 or anti-LFA-1 antibodies. (B) Quantification of T cell cluster formation in A calculated as the percentage of total area that is occupied by T-cell clusters. Pooled data from at least two independent experiments are shown. (C) Representative microphotographs showing destruction of already-formed T cell clusters of CD45.1+ CD8+ activated T lymphocytes being subsequently treated for 24 h with isotype control, anti-ICAM-1 or anti-LFA-1 antibodies. (D) Quantification of T cell clusters was performed as in B . (E) Transmigration of activated CD45.1+ CD8+ T cells for 16 h across monolayers of IMLEC through serum-containing medium. The assays were performed in the presence of anti-ICAM-1 or anti-LFA-1 antibodies or control IgG. Data from five independent experiments are shown. (F) Percentages of CD8+ T-lymphocytes remaining in the upper well of Boyden chambers after transmigration assays as in E. Percentages of total area occupied by T-cell clusters was calculated using a manual region of interest (ROI) based on CD8+ signal. At least nine pictures where analyzed in each case. (G) Representative confocal images of OT1 CD8+ lymphocytes (red) that remained in the upper well of transwell Boyden chambers after transmigration experiments as in E (* p
    Figure Legend Snippet: In vitro blockade of the ICAM-1/LFA-1 pair abolishes T cell clustering and speeds their transmigration across lymphatic monolayers. (A) Representative microphotographs of CD45.1+ CD8+ T cell clusters after in vitro activation for 24 or 48 h in the presence of control isotype or anti-ICAM-1 or anti-LFA-1 antibodies. (B) Quantification of T cell cluster formation in A calculated as the percentage of total area that is occupied by T-cell clusters. Pooled data from at least two independent experiments are shown. (C) Representative microphotographs showing destruction of already-formed T cell clusters of CD45.1+ CD8+ activated T lymphocytes being subsequently treated for 24 h with isotype control, anti-ICAM-1 or anti-LFA-1 antibodies. (D) Quantification of T cell clusters was performed as in B . (E) Transmigration of activated CD45.1+ CD8+ T cells for 16 h across monolayers of IMLEC through serum-containing medium. The assays were performed in the presence of anti-ICAM-1 or anti-LFA-1 antibodies or control IgG. Data from five independent experiments are shown. (F) Percentages of CD8+ T-lymphocytes remaining in the upper well of Boyden chambers after transmigration assays as in E. Percentages of total area occupied by T-cell clusters was calculated using a manual region of interest (ROI) based on CD8+ signal. At least nine pictures where analyzed in each case. (G) Representative confocal images of OT1 CD8+ lymphocytes (red) that remained in the upper well of transwell Boyden chambers after transmigration experiments as in E (* p

    Techniques Used: In Vitro, Transmigration Assay, Activation Assay

    In vivo activated T-cells experience increased migration from tumors to the LNs after ICAM-1 blockade. (A) Treatment schedule. (B) Percentages of intravenously injected CD45.1+ CD8+ T cells in the draining LNs 48 h after treatment with isotype ( n = 9) or anti-ICAM-1 ( n = 6) antibodies of mice bearing B16-VEGFC-OVA tumors. Cells were detected by flow cytometry. (C) Percentages of intravenously injected CD45.1+ CD8+ T cells that remained in B16-VEGFC-OVA tumors 48 h after treatment with isotype ( n = 9) or anti-ICAM-1 ( n = 6) antibodies and detected by flow cytometry. (D) Mean fluorescence intensity of CCR7 expression in CD45.1+ CD8+ T cells remaining in B16-VEGFC-OVA tumors in mice treated as in C. (* p
    Figure Legend Snippet: In vivo activated T-cells experience increased migration from tumors to the LNs after ICAM-1 blockade. (A) Treatment schedule. (B) Percentages of intravenously injected CD45.1+ CD8+ T cells in the draining LNs 48 h after treatment with isotype ( n = 9) or anti-ICAM-1 ( n = 6) antibodies of mice bearing B16-VEGFC-OVA tumors. Cells were detected by flow cytometry. (C) Percentages of intravenously injected CD45.1+ CD8+ T cells that remained in B16-VEGFC-OVA tumors 48 h after treatment with isotype ( n = 9) or anti-ICAM-1 ( n = 6) antibodies and detected by flow cytometry. (D) Mean fluorescence intensity of CCR7 expression in CD45.1+ CD8+ T cells remaining in B16-VEGFC-OVA tumors in mice treated as in C. (* p

    Techniques Used: In Vivo, Migration, Injection, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Expressing

    Integrin ligand blockade leads to increments in chemokine-dependent migration. (A) Surface expression of the chemokine receptor CCR7 on in vitro activated OT-1 CD8+ T cells in the presence or in the absence of anti-ICAM-1 antibodies. Flow-cytometry data was collected from three or more independent experiments. (B) Chemokine-directed migration of activated CD45.1+ CD8+ T lymphocytes in 3D collagen gels after being treated with anti-ICAM-1 antibodies or control IgG for 48 h. Cell movement toward CCL21 gradients was recorded by in vivo confocal microscopy and cell straightness was analyzed using IMARIS software. Each dot represents a single tracked cell. At least 80 cells were tracked in each case. (C) Percentages of intratumorally-injected CD45.1+ CD8+ T cells that reached the draining LNs 48 h after treatment with isotype antibody ( n = 35), isotype + PTx ( n = 6), anti-ICAM-1 antibody ( n = 39), or anti-ICAM-1 + PTx ( n = 10). (D) Mean fluorescence intensity of CCR7 expression in LN CD45.1+ CD8+ T cells 48 h after treatment with isotype ( n = 26) or anti-ICAM-1 ( n = 33) antibodies. Cells were detected by flow cytometry (** p
    Figure Legend Snippet: Integrin ligand blockade leads to increments in chemokine-dependent migration. (A) Surface expression of the chemokine receptor CCR7 on in vitro activated OT-1 CD8+ T cells in the presence or in the absence of anti-ICAM-1 antibodies. Flow-cytometry data was collected from three or more independent experiments. (B) Chemokine-directed migration of activated CD45.1+ CD8+ T lymphocytes in 3D collagen gels after being treated with anti-ICAM-1 antibodies or control IgG for 48 h. Cell movement toward CCL21 gradients was recorded by in vivo confocal microscopy and cell straightness was analyzed using IMARIS software. Each dot represents a single tracked cell. At least 80 cells were tracked in each case. (C) Percentages of intratumorally-injected CD45.1+ CD8+ T cells that reached the draining LNs 48 h after treatment with isotype antibody ( n = 35), isotype + PTx ( n = 6), anti-ICAM-1 antibody ( n = 39), or anti-ICAM-1 + PTx ( n = 10). (D) Mean fluorescence intensity of CCR7 expression in LN CD45.1+ CD8+ T cells 48 h after treatment with isotype ( n = 26) or anti-ICAM-1 ( n = 33) antibodies. Cells were detected by flow cytometry (** p

    Techniques Used: Migration, Expressing, In Vitro, Flow Cytometry, Cytometry, In Vivo, Confocal Microscopy, Software, Injection, Fluorescence

    Intratumoral CD8+ T-cells form diffuse cell-clusters that are reduced upon anti-ICAM-1 treatment. (A) Representative confocal images of B16-VEGFC tumors from isotype or anti-ICAM-1 treated mice showing CD45.1+ T cells (orange), CD8+ T cells (red), and intratumoral LVs (green). Images at the right end show magnifications of T cell clusters. (B) Percentages of area occupied by CD45.1+ CD8+ T-lymphocyte clusters in B16-VEGFC tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 13 pictures where analyzed in each case. (C) Representative confocal images of B16-VEGFC tumors from isotype or anti-LFA-1 treated mice showing CD45.1+ T cells (orange) and CD8+ T cells (red). (D) Percentages of area occupied by CD45.1+ CD8+ T-lymphocyte clusters in B16-VEGFC tumors 48 h after isotype or anti-LFA-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 19 pictures where analyzed in each case. (E) Representative confocal images of Her2/Neu tumors in isotype or anti-ICAM-1 treated mice showing CD8+ T cells (red). (F) Percentages of area occupied by CD8+ T-lymphocyte clusters in Her2/Neu tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 26 pictures where analyzed in each case. (G) Representative microphotograph showing CD8+ T cell clusters as detected by immunohistochemistry on samples obtained from a series of 10 human melanoma patients. Magnifications show two instances of T cell clusters (* p
    Figure Legend Snippet: Intratumoral CD8+ T-cells form diffuse cell-clusters that are reduced upon anti-ICAM-1 treatment. (A) Representative confocal images of B16-VEGFC tumors from isotype or anti-ICAM-1 treated mice showing CD45.1+ T cells (orange), CD8+ T cells (red), and intratumoral LVs (green). Images at the right end show magnifications of T cell clusters. (B) Percentages of area occupied by CD45.1+ CD8+ T-lymphocyte clusters in B16-VEGFC tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 13 pictures where analyzed in each case. (C) Representative confocal images of B16-VEGFC tumors from isotype or anti-LFA-1 treated mice showing CD45.1+ T cells (orange) and CD8+ T cells (red). (D) Percentages of area occupied by CD45.1+ CD8+ T-lymphocyte clusters in B16-VEGFC tumors 48 h after isotype or anti-LFA-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 19 pictures where analyzed in each case. (E) Representative confocal images of Her2/Neu tumors in isotype or anti-ICAM-1 treated mice showing CD8+ T cells (red). (F) Percentages of area occupied by CD8+ T-lymphocyte clusters in Her2/Neu tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of total area occupied by T cell clusters was calculated using a manual quantification of aggregates of four or more cells. At least 26 pictures where analyzed in each case. (G) Representative microphotograph showing CD8+ T cell clusters as detected by immunohistochemistry on samples obtained from a series of 10 human melanoma patients. Magnifications show two instances of T cell clusters (* p

    Techniques Used: Mouse Assay, Immunohistochemistry

    ICAM-1-LFA-1 dependent CD8+ T-lymphocyte aggregation in tumor tissue prevents recirculation to draining lymph nodes. (A) Intra-tumoral CD8+ T lymphocytes form clusters that are retained in the tumor microenvironment by homotypic ICAM-1-LFA-1 interactions. (B) ICAM-1 blockade results in destruction of T-cell clusters and increased T cell sensitivity to CCL21 chemokine-guided migration toward the lymphatic vessels, thus facilitating their transit to the draining lymph nodes.
    Figure Legend Snippet: ICAM-1-LFA-1 dependent CD8+ T-lymphocyte aggregation in tumor tissue prevents recirculation to draining lymph nodes. (A) Intra-tumoral CD8+ T lymphocytes form clusters that are retained in the tumor microenvironment by homotypic ICAM-1-LFA-1 interactions. (B) ICAM-1 blockade results in destruction of T-cell clusters and increased T cell sensitivity to CCL21 chemokine-guided migration toward the lymphatic vessels, thus facilitating their transit to the draining lymph nodes.

    Techniques Used: Migration

    Systemic ICAM-1 blockade increases the egress of intratumorally injected CD8+ T-cells to draining LNs. (A) Percentages of total CD45.1+ CD8+ T cells injected in B16-VEGFC tumors that reached the draining LNs 48 h after isotype ( n = 29), anti-ICAM-1 ( n = 32), or anti-LFA-1 ( n = 12) treatment and detected by flow cytometry. (B) Representative confocal images of B16-VEGFC tumors in mice treated with isotype, anti-ICAM-1 or anti-LFA-1 antibodies showing CD45.1+ T cells (red) and intratumoral LVs (green). (C) Percentages of CD45.1+ CD8+ T-lymphocytes remaining in B16-VEGFC tumors 48 h after isotype, anti-ICAM-1 or anti-LFA-1 antibody treatment. Percentage of positive area was calculated using a manual region of interest (ROI) based on CD45.1 signal. At least 12 pictures from 3 different tumors where analyzed in each case. (D) Percentages of total CD8+ T cells injected in Her2/Neu tumors that reached the draining LNs after isotype ( n = 6) or anti-ICAM-1 ( n = 8) treatment and detected by flow cytometry. (E) Percentages of total CD8+ T cells injected in Her2/Neu tumors that remained in tumors 48 h after isotype ( n = 6) or anti-ICAM-1 ( n = 8) treatment and detected by flow cytometry. (F) Percentages of CD8+ T-lymphocytes remaining in Her2/Neu tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of positive area was calculated using a manual region of interest (ROI) based on CD8 signal. At least 23 pictures from 3 different tumors where analyzed in each case (* p
    Figure Legend Snippet: Systemic ICAM-1 blockade increases the egress of intratumorally injected CD8+ T-cells to draining LNs. (A) Percentages of total CD45.1+ CD8+ T cells injected in B16-VEGFC tumors that reached the draining LNs 48 h after isotype ( n = 29), anti-ICAM-1 ( n = 32), or anti-LFA-1 ( n = 12) treatment and detected by flow cytometry. (B) Representative confocal images of B16-VEGFC tumors in mice treated with isotype, anti-ICAM-1 or anti-LFA-1 antibodies showing CD45.1+ T cells (red) and intratumoral LVs (green). (C) Percentages of CD45.1+ CD8+ T-lymphocytes remaining in B16-VEGFC tumors 48 h after isotype, anti-ICAM-1 or anti-LFA-1 antibody treatment. Percentage of positive area was calculated using a manual region of interest (ROI) based on CD45.1 signal. At least 12 pictures from 3 different tumors where analyzed in each case. (D) Percentages of total CD8+ T cells injected in Her2/Neu tumors that reached the draining LNs after isotype ( n = 6) or anti-ICAM-1 ( n = 8) treatment and detected by flow cytometry. (E) Percentages of total CD8+ T cells injected in Her2/Neu tumors that remained in tumors 48 h after isotype ( n = 6) or anti-ICAM-1 ( n = 8) treatment and detected by flow cytometry. (F) Percentages of CD8+ T-lymphocytes remaining in Her2/Neu tumors 48 h after isotype or anti-ICAM-1 antibody treatment. Percentage of positive area was calculated using a manual region of interest (ROI) based on CD8 signal. At least 23 pictures from 3 different tumors where analyzed in each case (* p

    Techniques Used: Injection, Flow Cytometry, Cytometry, Mouse Assay

    9) Product Images from "Assessment of ICAM-1 N-glycoforms in mouse and human models of endothelial dysfunction"

    Article Title: Assessment of ICAM-1 N-glycoforms in mouse and human models of endothelial dysfunction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0230358

    HM epitopes co-localize with ICAM-1 in high fat-induced mouse atherosclerosis. Total, HM / hybrid, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 were measured in the innominate and left carotid arteries from ApoE-/- mice fed a normal or high fat diet. A) Shown are representative images of innominate arteries from paired lesion and non-lesion areas of the same vessel section. Red staining represents total ICAM-1, red puncta represent positive PLA staining for specific ICAM-1 N-glycoforms (indicated by arrows), and blue staining represents DAPI. # indicates the lumen of each vessel. Panels B and C show PLA staining of lesion areas when the anti-ICAM-1 antibody or avidin were excluded. Panel D shows total ICAM-1 staining in lesion versus non-lesion areas. *p
    Figure Legend Snippet: HM epitopes co-localize with ICAM-1 in high fat-induced mouse atherosclerosis. Total, HM / hybrid, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 were measured in the innominate and left carotid arteries from ApoE-/- mice fed a normal or high fat diet. A) Shown are representative images of innominate arteries from paired lesion and non-lesion areas of the same vessel section. Red staining represents total ICAM-1, red puncta represent positive PLA staining for specific ICAM-1 N-glycoforms (indicated by arrows), and blue staining represents DAPI. # indicates the lumen of each vessel. Panels B and C show PLA staining of lesion areas when the anti-ICAM-1 antibody or avidin were excluded. Panel D shows total ICAM-1 staining in lesion versus non-lesion areas. *p

    Techniques Used: Mouse Assay, Staining, Proximity Ligation Assay, Avidin-Biotin Assay

    HM / hybrid, HM, α-2,6-sialylated, ICAM-1 are increased CKD patients with failed arteriovenous fistulas. Panel A shows total ICAM-1 (red staining) and specified N-glycoforms in vessels from CKD patients with failed or successful AVF creation. Red puncta represent positive PLA staining (as indicated by arrows). B) Quantification of total ICAM-1 signal in failed and successful AVF samples (n = 4 each). Error bars are mean ± SEM; * p
    Figure Legend Snippet: HM / hybrid, HM, α-2,6-sialylated, ICAM-1 are increased CKD patients with failed arteriovenous fistulas. Panel A shows total ICAM-1 (red staining) and specified N-glycoforms in vessels from CKD patients with failed or successful AVF creation. Red puncta represent positive PLA staining (as indicated by arrows). B) Quantification of total ICAM-1 signal in failed and successful AVF samples (n = 4 each). Error bars are mean ± SEM; * p

    Techniques Used: Staining, Proximity Ligation Assay

    CD68 macrophage staining positively correlates with HM-ICAM-1 in animal models of atherosclerosis. A-C. CD68 staining (fluorescence) as a function of ConA, HHL, and SNA puncta, respectively, for ApoE-/- mice fed HFD diet. n = 6 animals; n = 12 paired lesion and non-lesion vessel areas. D-F . CD68 staining (fluorescence) as a function of ConA, HHL, and SNA puncta, respectively, for ApoE-/- mice after partial carotid ligation n = 3 animals; n = 6 vessel samples. Best fit lines determined by linear regression with Pearson correlation analyses with indicated coefficients and p-values shown in each panel.
    Figure Legend Snippet: CD68 macrophage staining positively correlates with HM-ICAM-1 in animal models of atherosclerosis. A-C. CD68 staining (fluorescence) as a function of ConA, HHL, and SNA puncta, respectively, for ApoE-/- mice fed HFD diet. n = 6 animals; n = 12 paired lesion and non-lesion vessel areas. D-F . CD68 staining (fluorescence) as a function of ConA, HHL, and SNA puncta, respectively, for ApoE-/- mice after partial carotid ligation n = 3 animals; n = 6 vessel samples. Best fit lines determined by linear regression with Pearson correlation analyses with indicated coefficients and p-values shown in each panel.

    Techniques Used: Staining, Fluorescence, Mouse Assay, Ligation

    HM / hybrid, HM, and α-2,6-sialylated ICAM-1 are increased in mouse atherosclerosis after induction of disturbed flow in vivo . Panel A shows representative images of total ICAM-1 HM / hybrid, HM, α2,6-sialylated, and α2,3-sialylated ICAM-1 in the left carotid artery (after partial ligation) and paired right carotid artery (control). Positive PLA puncta are indicated by arrows. Panels B C show total ICAM-1 staining in left versus right carotid artery and ICAM-1 N-glycoforms puncta, respectively. Data are mean ± SEM, each symbol represents an individual mouse, n = 3. *p
    Figure Legend Snippet: HM / hybrid, HM, and α-2,6-sialylated ICAM-1 are increased in mouse atherosclerosis after induction of disturbed flow in vivo . Panel A shows representative images of total ICAM-1 HM / hybrid, HM, α2,6-sialylated, and α2,3-sialylated ICAM-1 in the left carotid artery (after partial ligation) and paired right carotid artery (control). Positive PLA puncta are indicated by arrows. Panels B C show total ICAM-1 staining in left versus right carotid artery and ICAM-1 N-glycoforms puncta, respectively. Data are mean ± SEM, each symbol represents an individual mouse, n = 3. *p

    Techniques Used: In Vivo, Ligation, Proximity Ligation Assay, Staining

    HM / hybrid ICAM-1 is increased in human atherosclerosis. Panel A shows representative images of total ICAM-1 (red staining) and specified N-glycoforms in human vessels with lesions spanning types 1–5. Red puncta represent positive PLA staining (as indicated by arrows). Panel B shows the quantification of total ICAM-1 in early (1–2) and late (3–5) disease stages. Each symbol represents a different patient, with same symbol representing multiple vessels from the same patient. Data are mean ± SEM, n = 7–10. * p
    Figure Legend Snippet: HM / hybrid ICAM-1 is increased in human atherosclerosis. Panel A shows representative images of total ICAM-1 (red staining) and specified N-glycoforms in human vessels with lesions spanning types 1–5. Red puncta represent positive PLA staining (as indicated by arrows). Panel B shows the quantification of total ICAM-1 in early (1–2) and late (3–5) disease stages. Each symbol represents a different patient, with same symbol representing multiple vessels from the same patient. Data are mean ± SEM, n = 7–10. * p

    Techniques Used: Staining, Proximity Ligation Assay

    Proximity-ligation assay (PLA) schematic and lectin specificity. Biotinylated lectins with indicated specificities were added to first label specific sugars. Then, anti-ICAM-1 antibody and avidin, conjugated to complementary oligos, were added. When lectin-recognized sugar epitopes are less than 40 nm from anti-ICAM-1, the complementary oligos hybridize and amplify, producing red fluorescent puncta. Right panel shows N-glycan structures recognized by different lectins used (adapted from [ 19 ]. Man = Mannose; Gal = Galactose; GlcNac = N-acetylglucosamine; Sa = Sialic acid; R = varying N-glycan structures.
    Figure Legend Snippet: Proximity-ligation assay (PLA) schematic and lectin specificity. Biotinylated lectins with indicated specificities were added to first label specific sugars. Then, anti-ICAM-1 antibody and avidin, conjugated to complementary oligos, were added. When lectin-recognized sugar epitopes are less than 40 nm from anti-ICAM-1, the complementary oligos hybridize and amplify, producing red fluorescent puncta. Right panel shows N-glycan structures recognized by different lectins used (adapted from [ 19 ]. Man = Mannose; Gal = Galactose; GlcNac = N-acetylglucosamine; Sa = Sialic acid; R = varying N-glycan structures.

    Techniques Used: Proximity Ligation Assay, Avidin-Biotin Assay

    CD68 macrophage staining positively correlates with HM / hybrid ICAM-1 in human atherosclerosis. Panels A and B show representative images and quantitation, respectively, of CD68 staining in advanced and early type lesions, as well as IgG control staining. Data are mean ± SEM, n = 7–10 * p
    Figure Legend Snippet: CD68 macrophage staining positively correlates with HM / hybrid ICAM-1 in human atherosclerosis. Panels A and B show representative images and quantitation, respectively, of CD68 staining in advanced and early type lesions, as well as IgG control staining. Data are mean ± SEM, n = 7–10 * p

    Techniques Used: Staining, Quantitation Assay

    10) Product Images from "An inflammation-induced mechanism for leukocyte transmigration across lymphatic vessel endothelium"

    Article Title: An inflammation-induced mechanism for leukocyte transmigration across lymphatic vessel endothelium

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051759

    VCAM-1 and ICAM-1 are expressed on both luminal and basolateral surfaces of HDLECs. Cells were cultured on clear-membrane inserts and stimulated with TNF-α for 24 h before staining with antipodoplanin (red) and either anti–ICAM-1 or anti–VCAM-1 (blue) and analysis by confocal microscopy. Staining was performed either on HDLECs cultured (A) in the absence of MDDCs or (B) at 10 h after addition of Cell Tracker Green fluorescently labeled MDDCs. (A, top) Asterisks depict the axis through which individual LECs were imaged in z-section (bottom). Bars, 10 μm.
    Figure Legend Snippet: VCAM-1 and ICAM-1 are expressed on both luminal and basolateral surfaces of HDLECs. Cells were cultured on clear-membrane inserts and stimulated with TNF-α for 24 h before staining with antipodoplanin (red) and either anti–ICAM-1 or anti–VCAM-1 (blue) and analysis by confocal microscopy. Staining was performed either on HDLECs cultured (A) in the absence of MDDCs or (B) at 10 h after addition of Cell Tracker Green fluorescently labeled MDDCs. (A, top) Asterisks depict the axis through which individual LECs were imaged in z-section (bottom). Bars, 10 μm.

    Techniques Used: Cell Culture, Staining, Confocal Microscopy, Labeling

    MDDC adhesion to TNF-α–stimulated HDLECs is dependent on ICAM-1 and VCAM-1. Cell Tracker Green fluorescently labeled MDDCs were applied to TNF-α–stimulated HDLEC monolayers plated in 24-well plates, preincubated with either IgG control or ICAM-1 mAbs (15.2 or P2A4) or VCAM-1 mAbs (P8B1 or IGII), in triplicate. At 3 and 10 h, the numbers of adherent MDDCs were measured. Representative data from three independent experiments are shown and represent the mean ± SEM ( n = 3).
    Figure Legend Snippet: MDDC adhesion to TNF-α–stimulated HDLECs is dependent on ICAM-1 and VCAM-1. Cell Tracker Green fluorescently labeled MDDCs were applied to TNF-α–stimulated HDLEC monolayers plated in 24-well plates, preincubated with either IgG control or ICAM-1 mAbs (15.2 or P2A4) or VCAM-1 mAbs (P8B1 or IGII), in triplicate. At 3 and 10 h, the numbers of adherent MDDCs were measured. Representative data from three independent experiments are shown and represent the mean ± SEM ( n = 3).

    Techniques Used: Labeling

    Inflammatory cytokines up-regulate surface expression of ICAM-1, VCAM-1, and E-selectin in cultured HDLECs. Cells were cultured for 24 h in the presence of individual proinflammatory cytokines or chemokines before immunostaining for either (A) VCAM-1 or (B) ICAM-1 and quantitation by FACS analysis. Data represent the mean ± SEM ( n = 3). (C and D) Dot plots show VCAM-1 or ICAM-1 expression in HDLECs cultured in the presence or absence (control) of TNF-α and assessed by dual staining for podoplanin and CAMs. Note that all cells expressing CAMs are positive for podoplanin (top right quadrants). (E) Representative double immunofluorescence micrographs showing induction of CAMs and E-selectin (green) in podoplanin-positive (red) HDLECs, as indicated with nuclei counterstained for DAPI. Bar, 50 μm.
    Figure Legend Snippet: Inflammatory cytokines up-regulate surface expression of ICAM-1, VCAM-1, and E-selectin in cultured HDLECs. Cells were cultured for 24 h in the presence of individual proinflammatory cytokines or chemokines before immunostaining for either (A) VCAM-1 or (B) ICAM-1 and quantitation by FACS analysis. Data represent the mean ± SEM ( n = 3). (C and D) Dot plots show VCAM-1 or ICAM-1 expression in HDLECs cultured in the presence or absence (control) of TNF-α and assessed by dual staining for podoplanin and CAMs. Note that all cells expressing CAMs are positive for podoplanin (top right quadrants). (E) Representative double immunofluorescence micrographs showing induction of CAMs and E-selectin (green) in podoplanin-positive (red) HDLECs, as indicated with nuclei counterstained for DAPI. Bar, 50 μm.

    Techniques Used: Expressing, Cell Culture, Immunostaining, Quantitation Assay, FACS, Staining, Immunofluorescence

    MDDC transmigration of TNF-α–stimulated HDLEC monolayers is dependent on ICAM-1 and VCAM-1. Transmigration of Cell Tracker Green fluorescently labeled MDDCs across either unstimulated or TNF-α–stimulated HDLEC monolayers plated on the undersurface of Fluoroblok filters was monitored in the presence or absence of selected adhesion blocking antibodies over a 12-h period. Progress curves are shown for MDDC transmigration across (A) control unstimulated versus TNF-α–stimulated HDLECs, (B) TNF-α–stimulated HDLECs treated with control rat IgG versus VCAM-1–neutralizing mAb P8B1, and (C) TNF-α–stimulated HDLECs treated with control rat IgG versus ICAM-1– neutralizing mAb P2A4. Data represent the mean ± SEM ( n = 4). (D) Comparative effects of individual ICAM-1 mAbs 15.2 and P2A4, VCAM-1 mAbs 51-10C9 and P8B1, ICAM-1 mAb 15.2 and VCAM-1 mAb P8B1 together, the LFA-1 mAb 24, and control mouse IgG on MDDC transmigration of TNF-α–stimulated HDLECs. The level of transmigration across unstimulated HDLECs is shown for comparison. Data from three independent experiments are normalized to the measured levels of transmigration in the presence of control IgG (100% maximal transmigration) in each case and represent the mean ± SEM ( n = 4). (E) Permeability of confluent HDLEC monolayers to unconjugated Alexa Fluor 488 measured as dye recovered in the lower chamber of Fluoroblok filter wells after a 6-h incubation at 37°C. Data represent the mean ± SEM.
    Figure Legend Snippet: MDDC transmigration of TNF-α–stimulated HDLEC monolayers is dependent on ICAM-1 and VCAM-1. Transmigration of Cell Tracker Green fluorescently labeled MDDCs across either unstimulated or TNF-α–stimulated HDLEC monolayers plated on the undersurface of Fluoroblok filters was monitored in the presence or absence of selected adhesion blocking antibodies over a 12-h period. Progress curves are shown for MDDC transmigration across (A) control unstimulated versus TNF-α–stimulated HDLECs, (B) TNF-α–stimulated HDLECs treated with control rat IgG versus VCAM-1–neutralizing mAb P8B1, and (C) TNF-α–stimulated HDLECs treated with control rat IgG versus ICAM-1– neutralizing mAb P2A4. Data represent the mean ± SEM ( n = 4). (D) Comparative effects of individual ICAM-1 mAbs 15.2 and P2A4, VCAM-1 mAbs 51-10C9 and P8B1, ICAM-1 mAb 15.2 and VCAM-1 mAb P8B1 together, the LFA-1 mAb 24, and control mouse IgG on MDDC transmigration of TNF-α–stimulated HDLECs. The level of transmigration across unstimulated HDLECs is shown for comparison. Data from three independent experiments are normalized to the measured levels of transmigration in the presence of control IgG (100% maximal transmigration) in each case and represent the mean ± SEM ( n = 4). (E) Permeability of confluent HDLEC monolayers to unconjugated Alexa Fluor 488 measured as dye recovered in the lower chamber of Fluoroblok filter wells after a 6-h incubation at 37°C. Data represent the mean ± SEM.

    Techniques Used: Transmigration Assay, Labeling, Blocking Assay, Permeability, Incubation

    In vivo expression of ICAM-1 and VCAM-1 in mouse dermal lymphatics induced by skin contact hypersensitivity. Skin inflammation was induced in mouse ear by sensitization and subsequent challenge with oxazolone before analysis of lymphatic vessel CAM expression by immunofluorescence microscopy. (A and B) Whole-mount sections of oxazolone-challenged and contralateral-unchallenged (control) ears dual-stained for podoplanin (green) and ICAM-1 or VCAM-1, respectively (red). Note the weak expression of ICAM-1 confined to podoplanin-negative (blood) vessels in uninflamed skin (A) and the focal up-regulation of both ICAM-1 and VCAM-1 on podoplanin-positive (lymphatic) vessels in inflamed skin (A and B). Images were captured by confocal microscopy. Bars, 100 μm. (C and D) Quantitative estimates for the numbers of ICAM-1 + /podoplanin + and VCAM-1 + /podoplanin + vessels determined by counting 21 separate fields of view (7 fields/mouse) in control and oxazolone-treated ear sections. Data represent the mean ± SEM.
    Figure Legend Snippet: In vivo expression of ICAM-1 and VCAM-1 in mouse dermal lymphatics induced by skin contact hypersensitivity. Skin inflammation was induced in mouse ear by sensitization and subsequent challenge with oxazolone before analysis of lymphatic vessel CAM expression by immunofluorescence microscopy. (A and B) Whole-mount sections of oxazolone-challenged and contralateral-unchallenged (control) ears dual-stained for podoplanin (green) and ICAM-1 or VCAM-1, respectively (red). Note the weak expression of ICAM-1 confined to podoplanin-negative (blood) vessels in uninflamed skin (A) and the focal up-regulation of both ICAM-1 and VCAM-1 on podoplanin-positive (lymphatic) vessels in inflamed skin (A and B). Images were captured by confocal microscopy. Bars, 100 μm. (C and D) Quantitative estimates for the numbers of ICAM-1 + /podoplanin + and VCAM-1 + /podoplanin + vessels determined by counting 21 separate fields of view (7 fields/mouse) in control and oxazolone-treated ear sections. Data represent the mean ± SEM.

    Techniques Used: In Vivo, Expressing, Chick Chorioallantoic Membrane Assay, Immunofluorescence, Microscopy, Staining, Confocal Microscopy

    Kinetics of TNF-induced CAM and E-selectin expression in cultured primary HDLECs. (A–C) Respective time courses for induction of VCAM-1, ICAM-1, and E-selectin in HDLECs cultured for 0–48 h in the presence or absence of 1 ng/ml TNF-α, as assessed by FACS analysis. Representative histograms are shown for cells stained with isotype-matched control Ig (light gray) or mAbs to the appropriate adhesion molecules in unstimulated cells (black) or cells treated with TNF-α for 3 h (dark gray), 6 h (blue), 12 h (green), and 24 h (red).
    Figure Legend Snippet: Kinetics of TNF-induced CAM and E-selectin expression in cultured primary HDLECs. (A–C) Respective time courses for induction of VCAM-1, ICAM-1, and E-selectin in HDLECs cultured for 0–48 h in the presence or absence of 1 ng/ml TNF-α, as assessed by FACS analysis. Representative histograms are shown for cells stained with isotype-matched control Ig (light gray) or mAbs to the appropriate adhesion molecules in unstimulated cells (black) or cells treated with TNF-α for 3 h (dark gray), 6 h (blue), 12 h (green), and 24 h (red).

    Techniques Used: Chick Chorioallantoic Membrane Assay, Expressing, Cell Culture, FACS, Staining

    In vivo trafficking of skin DCs via afferent lymphatics is dependent on ICAM-1 and VCAM-1 adhesion. The involvement of ICAM-1 and VCAM-1 in the trafficking of DCs via afferent lymphatics was investigated in mice with oxazolone-induced skin hypersensitivity. (A) Recoveries of FITC + /CD11c + skin DCs in the draining lymph nodes 24 h after FITC skin painting of oxazolone-sensitized mice that received prior injection of neutralizing mAbs to VCAM-1, ICAM-1, or control rat Ig. Data represent the mean recoveries ± SEM (obtained from three separate experiments). (B) To show retention of DCs within the skin, CMFDA-labeled bone marrow–derived DCs from a littermate were intradermally injected into the ear tissue of sensitized mice that received prior injection of a neutralizing mAb to ICAM-1 (YN1-1) or control rat Ig. After 24 h, ears were removed, and whole-mount staining was performed using antipodoplanin with Alexa Fluor 568 (red) and Cy5-conjugated goat anti–rat Cy5 (blue) to detect binding of neutralizing antibody within the tissue. Bars, 100 μm.
    Figure Legend Snippet: In vivo trafficking of skin DCs via afferent lymphatics is dependent on ICAM-1 and VCAM-1 adhesion. The involvement of ICAM-1 and VCAM-1 in the trafficking of DCs via afferent lymphatics was investigated in mice with oxazolone-induced skin hypersensitivity. (A) Recoveries of FITC + /CD11c + skin DCs in the draining lymph nodes 24 h after FITC skin painting of oxazolone-sensitized mice that received prior injection of neutralizing mAbs to VCAM-1, ICAM-1, or control rat Ig. Data represent the mean recoveries ± SEM (obtained from three separate experiments). (B) To show retention of DCs within the skin, CMFDA-labeled bone marrow–derived DCs from a littermate were intradermally injected into the ear tissue of sensitized mice that received prior injection of a neutralizing mAb to ICAM-1 (YN1-1) or control rat Ig. After 24 h, ears were removed, and whole-mount staining was performed using antipodoplanin with Alexa Fluor 568 (red) and Cy5-conjugated goat anti–rat Cy5 (blue) to detect binding of neutralizing antibody within the tissue. Bars, 100 μm.

    Techniques Used: In Vivo, Mouse Assay, Injection, Labeling, Derivative Assay, Staining, Binding Assay

    Related Articles

    Western Blot:

    Article Title: The Transcription Factor Erg Controls Endothelial Cell Quiescence by Repressing Activity of Nuclear Factor (NF)-?B p65 *
    Article Snippet: .. After 39 h, cells were treated with BAY 11-7085 (5 μm , Sigma) diluted in dimethyl sulfoxide and incubated for a further 24 h. Cells were treated with 10 ng/ml of TNF-α for the final 6 h. ICAM-1 protein levels were measured by Western blot using an anti-ICAM-1 (clone 15.2, kind gift of Prof. Nancy Hogg) antibody, and normalized to GAPDH (MAB374 Millipore). .. Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed using ChIP-IT express (Active Motif, Rixensart, Belgium) as previously described ( ).

    Incubation:

    Article Title: The Transcription Factor Erg Controls Endothelial Cell Quiescence by Repressing Activity of Nuclear Factor (NF)-?B p65 *
    Article Snippet: .. After 39 h, cells were treated with BAY 11-7085 (5 μm , Sigma) diluted in dimethyl sulfoxide and incubated for a further 24 h. Cells were treated with 10 ng/ml of TNF-α for the final 6 h. ICAM-1 protein levels were measured by Western blot using an anti-ICAM-1 (clone 15.2, kind gift of Prof. Nancy Hogg) antibody, and normalized to GAPDH (MAB374 Millipore). .. Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed using ChIP-IT express (Active Motif, Rixensart, Belgium) as previously described ( ).

    In Situ Hybridization:

    Article Title: Upregulation of ICAM-1 in diabetic rats after transient forebrain ischemia and reperfusion injury
    Article Snippet: .. Polyclonal anti-GFAP antibody (Santa Cruz), monoclonal anti-NeuN antibody (Sigma), polyclonal anti-ICAM-1 antibody (Protect), polyclonal anti-β-actin antibody (Sigma), horseradish peroxidase-conjugated anti-mouse secondary antibody (Sigma), and streptozotocin (STZ, Calbiochem, Germany) and the ICAM-1 In Situ Hybridization Detection kit were purchased from Boster Biotechnology Co (Wuhan, China). .. STZ-induced diabetic hyperglycemia The rats were injected intraperitoneally with streptozotocin (STZ, 55 mg/kg, in 0.1 mol/l citrate buffered saline, pH 4.5).

    Blocking Assay:

    Article Title: Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac–/ICAM-1-dependent mechanism
    Article Snippet: .. Mouse anti-human antibodies used in blocking studies were: anti-ICAM-1, clone P2A4 (Chemicon, Temecula, CA) and clone 15.2 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-LFA-1, clone TS1/22, (Pierce Biotechnology, Rockford, IL) and anti-Mac-1, clone CBRM1/5 (eBioscience, San Diego, CA). .. Isotype matched control used for blocking studies was mouse IgG1 from R & D Systems (Minneapolis, MN).

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  • 93
    Millipore anti icam 1
    Blocking adhesion through <t>ICAM-1</t> disrupts homotypic aggregation of HBZ-expressing Jurkat clones. (A) Effects of an ICAM-1-blocking antibody on homotypic aggregation. Upper panels, HBZ-expressing Jurkat clonal cells were plated at 5 × 10 5 cells/ml and cultured for 6 h prior to being photographed. Lower panels, HTLV-1-infected SLB-1 cells were plated at 1 × 10 6 cells/ml and cultured for 8.5 h prior to being photographed. At time zero, cellular aggregates were completely disrupted, and the antibodies indicated were added. The graph shows relative areas of aggregates averaged from three independent experiments, with the average aggregate area from the antibody-treated cells normalized to the average aggregate area from untreated cells (set to 100). The asterisk denotes a P value of
    Anti Icam 1, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore anti intercellular adhesion molecule icam 1
    A: NF-κB P-S276–p65 expression in ccRCC organ cultures. Untreated cultures (without TNF) show rare NF-κB P-S276–p65 expression in mTECs. In contrast, wtTNF- and R2-TNF-treated cultures show marked expression in mTECs ( arrows ). A positive signal but at a much lower level is detected in R1-TNF-treated cultures. Dual immunostaining show colocalization of <t>ICAM-1</t> (cytoplasm; green) and NF-κB P-S276–p65 (nuclei; red) in some tumor cells. Nuclei counterstained with To-PRO-3′ iodide. B: Analysis by paired Student’s t -test. Untreated versus R2-TNF or wtTNF and R2-TNF versus wtTNF: * P
    Anti Intercellular Adhesion Molecule Icam 1, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore mouse anti human icam 1
    Bilirubin suppresses cellular  ROS  generation following activation of  VCAM ‐1 or  ICAM ‐1.  TNF ‐α‐activated  HUVEC  monolayers were incubated with anti‐ VCAM ‐1 (left panels, α VCAM ‐1; 10 μg/mL) or anti‐ ICAM ‐1 (right panels, α ICAM ‐1; 10 μg/mL) for 30 minutes and then loaded with dihydrorhodamine. Adhesion molecule activation was triggered by the addition of a cross‐linking antibody and  ROS  generation quantified by confocal microscopy. Upper panels display representative time‐lapse images of nonstimulated and antibody‐activated cells treated with 20 μmol/L of bilirubin ( BR ) or the bilirubin vehicle (Veh). Scale bars represent 100 μm. Lower panels plot the time‐dependent changes in fluorescence intensity following  VCAM ‐1 (left panel) or  ICAM ‐1 (right panel) activation (squares), in the presence (white symbols) or absence (black symbols) of bilirubin. Cells that were not treated with cross‐linking antibodies (nonstimulated; circles) serve as control, with curves reflecting mean fluorescence intensity (±SEM) expressed relative to maximal activation at 60 minutes (n=3 sets of experiments). * P
    Mouse Anti Human Icam 1, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Blocking adhesion through ICAM-1 disrupts homotypic aggregation of HBZ-expressing Jurkat clones. (A) Effects of an ICAM-1-blocking antibody on homotypic aggregation. Upper panels, HBZ-expressing Jurkat clonal cells were plated at 5 × 10 5 cells/ml and cultured for 6 h prior to being photographed. Lower panels, HTLV-1-infected SLB-1 cells were plated at 1 × 10 6 cells/ml and cultured for 8.5 h prior to being photographed. At time zero, cellular aggregates were completely disrupted, and the antibodies indicated were added. The graph shows relative areas of aggregates averaged from three independent experiments, with the average aggregate area from the antibody-treated cells normalized to the average aggregate area from untreated cells (set to 100). The asterisk denotes a P value of

    Journal: Journal of Virology

    Article Title: Human T-Cell Leukemia Virus Type 1 (HTLV-1) bZIP Factor Upregulates the Expression of ICAM-1 To Facilitate HTLV-1 Infection

    doi: 10.1128/JVI.00608-19

    Figure Lengend Snippet: Blocking adhesion through ICAM-1 disrupts homotypic aggregation of HBZ-expressing Jurkat clones. (A) Effects of an ICAM-1-blocking antibody on homotypic aggregation. Upper panels, HBZ-expressing Jurkat clonal cells were plated at 5 × 10 5 cells/ml and cultured for 6 h prior to being photographed. Lower panels, HTLV-1-infected SLB-1 cells were plated at 1 × 10 6 cells/ml and cultured for 8.5 h prior to being photographed. At time zero, cellular aggregates were completely disrupted, and the antibodies indicated were added. The graph shows relative areas of aggregates averaged from three independent experiments, with the average aggregate area from the antibody-treated cells normalized to the average aggregate area from untreated cells (set to 100). The asterisk denotes a P value of

    Article Snippet: Blocking antibodies used in the experiments shown were as follows: anti-αM (M1/70; EMD Millipore), anti-αL (TS1/22; University of Iowa Developmental Studies Hybridoma Bank), anti-β2 (TS1/18; Thermo Fisher Scientific), anti-LFA-3 (TS2/9; Thermo Fisher Scientific), and anti-ICAM-1 (P2A4; EMD Millipore).

    Techniques: Blocking Assay, Expressing, Cell Culture, Infection

    Among the ICAM family members, HBZ-mediated activation is restricted to ICAM-1 and does not depend on JunD. (A) ICAM1 mRNA levels in empty-vector and HBZ-expressing HeLa clones. The graph shows qRT-PCR data averaged from three independent experiments, with data normalized to values for the empty-vector clone (set to 1). Mutations in the activation domain, leucine zipper domain, and the start codon of HBZ are indicated as mutAD, mutZIP, and mutATG, respectively; wt, wild type. Error bars show standard deviations. Significance was determined by a two-tailed Student's t test (***, P

    Journal: Journal of Virology

    Article Title: Human T-Cell Leukemia Virus Type 1 (HTLV-1) bZIP Factor Upregulates the Expression of ICAM-1 To Facilitate HTLV-1 Infection

    doi: 10.1128/JVI.00608-19

    Figure Lengend Snippet: Among the ICAM family members, HBZ-mediated activation is restricted to ICAM-1 and does not depend on JunD. (A) ICAM1 mRNA levels in empty-vector and HBZ-expressing HeLa clones. The graph shows qRT-PCR data averaged from three independent experiments, with data normalized to values for the empty-vector clone (set to 1). Mutations in the activation domain, leucine zipper domain, and the start codon of HBZ are indicated as mutAD, mutZIP, and mutATG, respectively; wt, wild type. Error bars show standard deviations. Significance was determined by a two-tailed Student's t test (***, P

    Article Snippet: Blocking antibodies used in the experiments shown were as follows: anti-αM (M1/70; EMD Millipore), anti-αL (TS1/22; University of Iowa Developmental Studies Hybridoma Bank), anti-β2 (TS1/18; Thermo Fisher Scientific), anti-LFA-3 (TS2/9; Thermo Fisher Scientific), and anti-ICAM-1 (P2A4; EMD Millipore).

    Techniques: Activation Assay, Plasmid Preparation, Expressing, Clone Assay, Quantitative RT-PCR, Two Tailed Test

    Jurkat cells with low HBZ expression maintain the ability to increase infection efficiency. (A) HBZ expression in Jurkat clone G6 is lower than in the other clones. Whole-cell extracts from the indicated Jurkat clonal cell lines were analyzed by Western blotting using an antibody against the C-terminal 6×His epitope tag (His) on HBZ. The membrane was stripped and reprobed with an antibody against actin. (B) Homotypic aggregation of the G6 clone. Cells were plated at 1 × 10 6 cells/ml, at which time cellular aggregates were completely disrupted. Cells were cultured for 3 h to allow aggregates to reform and then were photographed. (C) Cell surface abundance of ICAM-1 is elevated in Jurkat cells with low HBZ expression. Histograms show the relative cell surface abundance of ICAM-1 on the Jurkat empty-vector clones (yellow; C2, C4, and C5) and HBZ-expressing clones (blue; F10 and G6). Histograms for clones F10 and G6 are indicated by arrows. The gray histogram represents clone C2 cells probed with secondary antibody. (D) As effector cells, Jurkat cells with low HBZ expression produce higher levels of infection than the empty-vector clones. The indicated HBZ-expressing (light bars) and empty-vector (dark bars) Jurkat clones were transfected with pCMVHT1M-Tax9Q, pCRU5H-inLuc, and pSG-Tax. Clone F10 was separately cotransfected with pSG5 in place of pSG-Tax [F10 (-)]. Effector cells were cocultured with CHO-LFA-1 cells for 1.5 to 2 h and removed, and luciferase assays were performed on the CHO-LFA-1 cells 48 h later. The graph shows infection as a measure of relative luminescence units (RLUs) produced by the indicated effector cells, with data averaged from at least three replicates per treatment from a single experiment. The graph is representative of the results of two independent experiments. Error bars show standard deviations. Significance was determined by a two-tailed Student's t test (*, P

    Journal: Journal of Virology

    Article Title: Human T-Cell Leukemia Virus Type 1 (HTLV-1) bZIP Factor Upregulates the Expression of ICAM-1 To Facilitate HTLV-1 Infection

    doi: 10.1128/JVI.00608-19

    Figure Lengend Snippet: Jurkat cells with low HBZ expression maintain the ability to increase infection efficiency. (A) HBZ expression in Jurkat clone G6 is lower than in the other clones. Whole-cell extracts from the indicated Jurkat clonal cell lines were analyzed by Western blotting using an antibody against the C-terminal 6×His epitope tag (His) on HBZ. The membrane was stripped and reprobed with an antibody against actin. (B) Homotypic aggregation of the G6 clone. Cells were plated at 1 × 10 6 cells/ml, at which time cellular aggregates were completely disrupted. Cells were cultured for 3 h to allow aggregates to reform and then were photographed. (C) Cell surface abundance of ICAM-1 is elevated in Jurkat cells with low HBZ expression. Histograms show the relative cell surface abundance of ICAM-1 on the Jurkat empty-vector clones (yellow; C2, C4, and C5) and HBZ-expressing clones (blue; F10 and G6). Histograms for clones F10 and G6 are indicated by arrows. The gray histogram represents clone C2 cells probed with secondary antibody. (D) As effector cells, Jurkat cells with low HBZ expression produce higher levels of infection than the empty-vector clones. The indicated HBZ-expressing (light bars) and empty-vector (dark bars) Jurkat clones were transfected with pCMVHT1M-Tax9Q, pCRU5H-inLuc, and pSG-Tax. Clone F10 was separately cotransfected with pSG5 in place of pSG-Tax [F10 (-)]. Effector cells were cocultured with CHO-LFA-1 cells for 1.5 to 2 h and removed, and luciferase assays were performed on the CHO-LFA-1 cells 48 h later. The graph shows infection as a measure of relative luminescence units (RLUs) produced by the indicated effector cells, with data averaged from at least three replicates per treatment from a single experiment. The graph is representative of the results of two independent experiments. Error bars show standard deviations. Significance was determined by a two-tailed Student's t test (*, P

    Article Snippet: Blocking antibodies used in the experiments shown were as follows: anti-αM (M1/70; EMD Millipore), anti-αL (TS1/22; University of Iowa Developmental Studies Hybridoma Bank), anti-β2 (TS1/18; Thermo Fisher Scientific), anti-LFA-3 (TS2/9; Thermo Fisher Scientific), and anti-ICAM-1 (P2A4; EMD Millipore).

    Techniques: Expressing, Infection, Clone Assay, Western Blot, Cell Culture, Plasmid Preparation, Transfection, Luciferase, Produced, Two Tailed Test

    Levels of ICAM1 activation by HBZ and Tax are similar. (A) ICAM1 mRNA levels in the indicated T-cell lines. The graph shows qRT-PCR results averaged from two independent experiments in which values were normalized to those for the resting CD4 T cells (set to 1). Numbers above the bars indicate fold differences relative to the resting CD4 T cells. TL-Om1 cells (dark gray bar) express only HBZ, as the proviral sense strand-encoding genes are turned off. (B) ICAM1 mRNA levels from the empty-vector cells, HBZ-expressing HeLa clone, and Tax-transfected HeLa cells. The graph shows qRT-PCR data averaged from three independent experiments, with data normalized to values from cells carrying the empty vector (pcDNA or pSG5; set to 1). Error bars show standard deviations. Western blot analysis was performed using whole-cell extracts prepared from the empty-vector cells, HBZ-expressing HeLa clone, and Tax-transfected HeLa cells. Membranes were probed with an antibody against His to detect the C-terminal epitope tag on HBZ and Tax and then stripped and probed for actin. (C) Flow cytometry analysis of ICAM-1 on the empty-vector cells, HBZ-expressing HeLa clone, and Tax-transfected HeLa cells. Histograms on the left show relative cell surface levels of ICAM-1 on the HBZ-expressing clone, and histograms in the middle show relative cell surface levels of ICAM-1 on Tax-transfected cells (empty-vector cells, yellow; HBZ- and Tax-expressing cells, blue; isotype control antibody probing the HBZ- and Tax-expressing cells, gray). The bar graph on the right shows the fold change in the geometric mean fluorescence intensities (Geo-MFI) between empty-vector and HBZ- or Tax-expressing cells.

    Journal: Journal of Virology

    Article Title: Human T-Cell Leukemia Virus Type 1 (HTLV-1) bZIP Factor Upregulates the Expression of ICAM-1 To Facilitate HTLV-1 Infection

    doi: 10.1128/JVI.00608-19

    Figure Lengend Snippet: Levels of ICAM1 activation by HBZ and Tax are similar. (A) ICAM1 mRNA levels in the indicated T-cell lines. The graph shows qRT-PCR results averaged from two independent experiments in which values were normalized to those for the resting CD4 T cells (set to 1). Numbers above the bars indicate fold differences relative to the resting CD4 T cells. TL-Om1 cells (dark gray bar) express only HBZ, as the proviral sense strand-encoding genes are turned off. (B) ICAM1 mRNA levels from the empty-vector cells, HBZ-expressing HeLa clone, and Tax-transfected HeLa cells. The graph shows qRT-PCR data averaged from three independent experiments, with data normalized to values from cells carrying the empty vector (pcDNA or pSG5; set to 1). Error bars show standard deviations. Western blot analysis was performed using whole-cell extracts prepared from the empty-vector cells, HBZ-expressing HeLa clone, and Tax-transfected HeLa cells. Membranes were probed with an antibody against His to detect the C-terminal epitope tag on HBZ and Tax and then stripped and probed for actin. (C) Flow cytometry analysis of ICAM-1 on the empty-vector cells, HBZ-expressing HeLa clone, and Tax-transfected HeLa cells. Histograms on the left show relative cell surface levels of ICAM-1 on the HBZ-expressing clone, and histograms in the middle show relative cell surface levels of ICAM-1 on Tax-transfected cells (empty-vector cells, yellow; HBZ- and Tax-expressing cells, blue; isotype control antibody probing the HBZ- and Tax-expressing cells, gray). The bar graph on the right shows the fold change in the geometric mean fluorescence intensities (Geo-MFI) between empty-vector and HBZ- or Tax-expressing cells.

    Article Snippet: Blocking antibodies used in the experiments shown were as follows: anti-αM (M1/70; EMD Millipore), anti-αL (TS1/22; University of Iowa Developmental Studies Hybridoma Bank), anti-β2 (TS1/18; Thermo Fisher Scientific), anti-LFA-3 (TS2/9; Thermo Fisher Scientific), and anti-ICAM-1 (P2A4; EMD Millipore).

    Techniques: Activation Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Transfection, Western Blot, Flow Cytometry, Fluorescence

    HBZ upregulates ICAM-1 expression. (A) ICAM1 mRNA levels in empty-vector and HBZ-expressing Jurkat clones. The graph shows qRT-PCR data averaged from seven independent experiments, with data normalized to values for empty-vector clone C4 (set to 1). Error bars show standard deviations. Significance was determined by a one-way ANOVA comparing the clones ( P

    Journal: Journal of Virology

    Article Title: Human T-Cell Leukemia Virus Type 1 (HTLV-1) bZIP Factor Upregulates the Expression of ICAM-1 To Facilitate HTLV-1 Infection

    doi: 10.1128/JVI.00608-19

    Figure Lengend Snippet: HBZ upregulates ICAM-1 expression. (A) ICAM1 mRNA levels in empty-vector and HBZ-expressing Jurkat clones. The graph shows qRT-PCR data averaged from seven independent experiments, with data normalized to values for empty-vector clone C4 (set to 1). Error bars show standard deviations. Significance was determined by a one-way ANOVA comparing the clones ( P

    Article Snippet: Blocking antibodies used in the experiments shown were as follows: anti-αM (M1/70; EMD Millipore), anti-αL (TS1/22; University of Iowa Developmental Studies Hybridoma Bank), anti-β2 (TS1/18; Thermo Fisher Scientific), anti-LFA-3 (TS2/9; Thermo Fisher Scientific), and anti-ICAM-1 (P2A4; EMD Millipore).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Quantitative RT-PCR

    Effect of CD11 and CD18-blocking antibodies on the formation of clumps. A Semi-quantitative PCRs were performed to detect the expression of the ICAM-1 transcript. GAPDH is used as a DNA amount control. B A375, 1205LU and SLM8 cell lines were treated with 2 μg/ml of CD11a or CD18-blocking antibodies as indicated. Melanoma cells were labeled with DiO then fixed and labeled with DAPI prior to their observation under an epifluorescence microscope using a magnification of x10. Data were obtained from 3 independent experiments.

    Journal: BMC Cancer

    Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    doi: 10.1186/1471-2407-12-455

    Figure Lengend Snippet: Effect of CD11 and CD18-blocking antibodies on the formation of clumps. A Semi-quantitative PCRs were performed to detect the expression of the ICAM-1 transcript. GAPDH is used as a DNA amount control. B A375, 1205LU and SLM8 cell lines were treated with 2 μg/ml of CD11a or CD18-blocking antibodies as indicated. Melanoma cells were labeled with DiO then fixed and labeled with DAPI prior to their observation under an epifluorescence microscope using a magnification of x10. Data were obtained from 3 independent experiments.

    Article Snippet: 5×104 fluorescent melanoma cells were added to wells containing either 1μg/ml of isotypic control (IgG from BD Pharmingen, San Diego, CA, USA); or anti-CD11a (ab3981; Abcam; Paris, France), anti-CD18 (ab8220; Abcam; Paris, France) or anti-ICAM-1 (MAB2146Z; Millipore; Molsheim France) antibodies in the upper chamber.

    Techniques: Blocking Assay, Expressing, Labeling, Microscopy

    Conditioned mediums from melanoma cell lines enhance transcript expression of ICAM-1 in HUVEC cells. Semi-quantitative PCRs were performed to detect expression of ICAM-1 transcripts. A HUVEC cells were treated either with TNF-α and IFN-γ at 100ng/ml or B with conditioned medium from A375 (H+A375), SLM8 (H+SLM8) and 1205LU (H+1205LU) after 48hrs of cell culture. GAPDH is used as a DNA amount control. Data were obtained from 3 independent experiments.

    Journal: BMC Cancer

    Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    doi: 10.1186/1471-2407-12-455

    Figure Lengend Snippet: Conditioned mediums from melanoma cell lines enhance transcript expression of ICAM-1 in HUVEC cells. Semi-quantitative PCRs were performed to detect expression of ICAM-1 transcripts. A HUVEC cells were treated either with TNF-α and IFN-γ at 100ng/ml or B with conditioned medium from A375 (H+A375), SLM8 (H+SLM8) and 1205LU (H+1205LU) after 48hrs of cell culture. GAPDH is used as a DNA amount control. Data were obtained from 3 independent experiments.

    Article Snippet: 5×104 fluorescent melanoma cells were added to wells containing either 1μg/ml of isotypic control (IgG from BD Pharmingen, San Diego, CA, USA); or anti-CD11a (ab3981; Abcam; Paris, France), anti-CD18 (ab8220; Abcam; Paris, France) or anti-ICAM-1 (MAB2146Z; Millipore; Molsheim France) antibodies in the upper chamber.

    Techniques: Expressing, Cell Culture

    Effect of ICAM-1-blocking antibodies on the transendothelial migration of A375, 1205LU and SLM8 cell lines. The experiments were performed as detailed in Figure 1 , except that 2μg/ml of ICAM-1-blocking antibodies were introduced in the upper chamber of the Transwell when indicated. Histograms represent 3 independent experiments. In each experiment each condition was analyzed in duplicate.

    Journal: BMC Cancer

    Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    doi: 10.1186/1471-2407-12-455

    Figure Lengend Snippet: Effect of ICAM-1-blocking antibodies on the transendothelial migration of A375, 1205LU and SLM8 cell lines. The experiments were performed as detailed in Figure 1 , except that 2μg/ml of ICAM-1-blocking antibodies were introduced in the upper chamber of the Transwell when indicated. Histograms represent 3 independent experiments. In each experiment each condition was analyzed in duplicate.

    Article Snippet: 5×104 fluorescent melanoma cells were added to wells containing either 1μg/ml of isotypic control (IgG from BD Pharmingen, San Diego, CA, USA); or anti-CD11a (ab3981; Abcam; Paris, France), anti-CD18 (ab8220; Abcam; Paris, France) or anti-ICAM-1 (MAB2146Z; Millipore; Molsheim France) antibodies in the upper chamber.

    Techniques: Blocking Assay, Migration

    A: NF-κB P-S276–p65 expression in ccRCC organ cultures. Untreated cultures (without TNF) show rare NF-κB P-S276–p65 expression in mTECs. In contrast, wtTNF- and R2-TNF-treated cultures show marked expression in mTECs ( arrows ). A positive signal but at a much lower level is detected in R1-TNF-treated cultures. Dual immunostaining show colocalization of ICAM-1 (cytoplasm; green) and NF-κB P-S276–p65 (nuclei; red) in some tumor cells. Nuclei counterstained with To-PRO-3′ iodide. B: Analysis by paired Student’s t -test. Untreated versus R2-TNF or wtTNF and R2-TNF versus wtTNF: * P

    Journal: The American Journal of Pathology

    Article Title: Tumor Necrosis Factor Receptor Expression and Signaling in Renal Cell Carcinoma

    doi: 10.2353/ajpath.2010.091218

    Figure Lengend Snippet: A: NF-κB P-S276–p65 expression in ccRCC organ cultures. Untreated cultures (without TNF) show rare NF-κB P-S276–p65 expression in mTECs. In contrast, wtTNF- and R2-TNF-treated cultures show marked expression in mTECs ( arrows ). A positive signal but at a much lower level is detected in R1-TNF-treated cultures. Dual immunostaining show colocalization of ICAM-1 (cytoplasm; green) and NF-κB P-S276–p65 (nuclei; red) in some tumor cells. Nuclei counterstained with To-PRO-3′ iodide. B: Analysis by paired Student’s t -test. Untreated versus R2-TNF or wtTNF and R2-TNF versus wtTNF: * P

    Article Snippet: Antibodies used were goat anti-TNFR1, mouse anti-TNFR1, and mouse anti-TNFR2 (all from R & D Systems, Abington, UK), rabbit anti-TNFR2, rabbit anti-Etk/Bmx, and mouse anti-VEGF (all from Autogen Bioclear, Wiltshire, UK), mouse anti-RCC marker (RCC-Ma) and mouse anti-CD10 (both from Novocastra, Newcastle-on-Tyne, UK), mouse anti-pan-cytokeratin (pan-CK) and mouse anti-vimentin (both from DakoCytomation, Ely, UK), rabbit anti-ASK1-pSer967 or -pThr845 and anti-phospho-EtkpTyr40 , rabbit anti-cleaved active caspase-3p175 , and rabbit anti-NF-κBP-S276-p65 (all from Cell Signaling, Hertfordshire, UK), rabbit anti-phospho-VEGFR2pY1054-59 , rabbit anti-histone-H3phospho-S10 , and rabbit anti-ki67 (all from Abcam, Cambridge, UK), and anti-intercellular adhesion molecule (ICAM)-1 (Millipore, Watford, UK).

    Techniques: Expressing, Immunostaining

    Bilirubin suppresses cellular  ROS  generation following activation of  VCAM ‐1 or  ICAM ‐1.  TNF ‐α‐activated  HUVEC  monolayers were incubated with anti‐ VCAM ‐1 (left panels, α VCAM ‐1; 10 μg/mL) or anti‐ ICAM ‐1 (right panels, α ICAM ‐1; 10 μg/mL) for 30 minutes and then loaded with dihydrorhodamine. Adhesion molecule activation was triggered by the addition of a cross‐linking antibody and  ROS  generation quantified by confocal microscopy. Upper panels display representative time‐lapse images of nonstimulated and antibody‐activated cells treated with 20 μmol/L of bilirubin ( BR ) or the bilirubin vehicle (Veh). Scale bars represent 100 μm. Lower panels plot the time‐dependent changes in fluorescence intensity following  VCAM ‐1 (left panel) or  ICAM ‐1 (right panel) activation (squares), in the presence (white symbols) or absence (black symbols) of bilirubin. Cells that were not treated with cross‐linking antibodies (nonstimulated; circles) serve as control, with curves reflecting mean fluorescence intensity (±SEM) expressed relative to maximal activation at 60 minutes (n=3 sets of experiments). * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Bilirubin Prevents Atherosclerotic Lesion Formation in Low‐Density Lipoprotein Receptor‐Deficient Mice by Inhibiting Endothelial VCAM‐1 and ICAM‐1 Signaling

    doi: 10.1161/JAHA.116.004820

    Figure Lengend Snippet: Bilirubin suppresses cellular ROS generation following activation of VCAM ‐1 or ICAM ‐1. TNF ‐α‐activated HUVEC monolayers were incubated with anti‐ VCAM ‐1 (left panels, α VCAM ‐1; 10 μg/mL) or anti‐ ICAM ‐1 (right panels, α ICAM ‐1; 10 μg/mL) for 30 minutes and then loaded with dihydrorhodamine. Adhesion molecule activation was triggered by the addition of a cross‐linking antibody and ROS generation quantified by confocal microscopy. Upper panels display representative time‐lapse images of nonstimulated and antibody‐activated cells treated with 20 μmol/L of bilirubin ( BR ) or the bilirubin vehicle (Veh). Scale bars represent 100 μm. Lower panels plot the time‐dependent changes in fluorescence intensity following VCAM ‐1 (left panel) or ICAM ‐1 (right panel) activation (squares), in the presence (white symbols) or absence (black symbols) of bilirubin. Cells that were not treated with cross‐linking antibodies (nonstimulated; circles) serve as control, with curves reflecting mean fluorescence intensity (±SEM) expressed relative to maximal activation at 60 minutes (n=3 sets of experiments). * P

    Article Snippet: Mouse anti‐human VCAM‐1 (clone P3C4) and mouse anti‐human ICAM‐1 (clone P2A4) were purchased from Millipore (Temecula, CA).

    Techniques: Activation Assay, Incubation, Confocal Microscopy, Fluorescence

    Bilirubin does not alter adhesion molecule expression or monocyte binding to  HUVECs .  HUVEC  monolayers were incubated with or without  TNF ‐α (5 ng/mL) in the presence of bilirubin ( BR ; 20 μmol/L) or the bilirubin vehicle (Veh). A,  mRNA  levels for  ICAM ‐1,  VCAM ‐1,  PECAM ‐1, E‐Selectin, and P‐Selectin at baseline and at 4 hours (n=4 sets of experiments). The right lower panel depicts the results of an adhesion assay measuring the binding of CellTrace Far Red–labeled  THP ‐1 cells to  HUVEC  monolayers incubated with or without  TNF ‐α for 24 hours. Bars reflect mean fluorescence intensity (±SEM) relative to non‐ TNF ‐α‐activated  HUVECs  (n=4 sets of experiments). B, Representative immunoblots for E‐Selectin,  ICAM ‐1, and  VCAM ‐1, which are quantified (C) as described in Figure   1  (n=4 sets of experiments). * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Bilirubin Prevents Atherosclerotic Lesion Formation in Low‐Density Lipoprotein Receptor‐Deficient Mice by Inhibiting Endothelial VCAM‐1 and ICAM‐1 Signaling

    doi: 10.1161/JAHA.116.004820

    Figure Lengend Snippet: Bilirubin does not alter adhesion molecule expression or monocyte binding to HUVECs . HUVEC monolayers were incubated with or without TNF ‐α (5 ng/mL) in the presence of bilirubin ( BR ; 20 μmol/L) or the bilirubin vehicle (Veh). A, mRNA levels for ICAM ‐1, VCAM ‐1, PECAM ‐1, E‐Selectin, and P‐Selectin at baseline and at 4 hours (n=4 sets of experiments). The right lower panel depicts the results of an adhesion assay measuring the binding of CellTrace Far Red–labeled THP ‐1 cells to HUVEC monolayers incubated with or without TNF ‐α for 24 hours. Bars reflect mean fluorescence intensity (±SEM) relative to non‐ TNF ‐α‐activated HUVECs (n=4 sets of experiments). B, Representative immunoblots for E‐Selectin, ICAM ‐1, and VCAM ‐1, which are quantified (C) as described in Figure  1 (n=4 sets of experiments). * P

    Article Snippet: Mouse anti‐human VCAM‐1 (clone P3C4) and mouse anti‐human ICAM‐1 (clone P2A4) were purchased from Millipore (Temecula, CA).

    Techniques: Expressing, Binding Assay, Incubation, Cell Adhesion Assay, Labeling, Fluorescence, Western Blot

    Bilirubin does not alter VCAM ‐1 or ICAM ‐1 expression in Ldlr −/− mice. A and B, Representative photomicrographs of sections of aortic root stained with immunofluorescent antibodies against VCAM ‐1 (green; A) or ICAM ‐1 (cyan; B). Data are presented and analyzed as described in Figure 9 . DAPI indicates 4',6‐diamidino‐2‐phenylindole; ICAM‐1, intercellular adhesion molecule 1; VCAM‐1, vascular cell adhesion molecule 1.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Bilirubin Prevents Atherosclerotic Lesion Formation in Low‐Density Lipoprotein Receptor‐Deficient Mice by Inhibiting Endothelial VCAM‐1 and ICAM‐1 Signaling

    doi: 10.1161/JAHA.116.004820

    Figure Lengend Snippet: Bilirubin does not alter VCAM ‐1 or ICAM ‐1 expression in Ldlr −/− mice. A and B, Representative photomicrographs of sections of aortic root stained with immunofluorescent antibodies against VCAM ‐1 (green; A) or ICAM ‐1 (cyan; B). Data are presented and analyzed as described in Figure 9 . DAPI indicates 4',6‐diamidino‐2‐phenylindole; ICAM‐1, intercellular adhesion molecule 1; VCAM‐1, vascular cell adhesion molecule 1.

    Article Snippet: Mouse anti‐human VCAM‐1 (clone P3C4) and mouse anti‐human ICAM‐1 (clone P2A4) were purchased from Millipore (Temecula, CA).

    Techniques: Expressing, Mouse Assay, Staining

    Nox and  XO  inhibitors and antibodies against  VCAM ‐1 and  ICAM ‐1 recapitulate the effect of bilirubin on endothelial  ROS  generation and monocyte transmigration.  ROS  production by  TNF ‐α‐stimulated  HUVEC  monolayers was assessed by monitoring dihydrorhodamine fluorescence following activation of  VCAM ‐1 (α VCAM ‐1) or  ICAM ‐1 (α ICAM ‐1), as described in Figure   5 . A and B, Time‐dependent changes in fluorescence intensity following  VCAM ‐1 (A) or  ICAM ‐1 (B) activation (squares), in the absence (black symbols) or presence of 10 μmol/L of  ML 171 (white symbols), or 40 μmol/L of allopurinol ( AP ; gray symbols). Curves reflect mean fluorescence intensity (±SEM) expressed relative to maximal activation at 60 minutes (n=3 sets of experiments). C, Compares the effect of the  DMSO  vehicle (Veh), 40 μmol/L of  AP , and/or 10 μmol/L of  ML 171 on  THP ‐1 cell migration across  HUVEC  monolayers, as described in Figure   2  (n=4 sets of experiments). D and E, Results of analogous studies examining  THP ‐1 migration in the presence or absence of antibodies against  ICAM ‐1 ( ICAM ; 10 μg/mL),  VCAM ‐1 ( VCAM ; 10 μg/mL), β 2  (5 μg/mL), and/or α 4  (20 μg/mL). F, Lineweaver–Burk plot of H 2 O 2  produced by isolated XO in the presence of 50 μmol/L of bilirubin ( BR ; diamonds; K i =3.4 μmol/L), 30 μmol/L of the competitive inhibitor, AP (triangles; K i =6.7 μmol/L), 30 μmol/L of the noncompetitive inhibitor, 2‐chloro‐6(methylamino) purine ( CMAP ; squares; K i =4.7 μmol/L), or the  DMSO  vehicle (circles). Data reflect the mean (±SEM) of 3 sets of experiments. * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Bilirubin Prevents Atherosclerotic Lesion Formation in Low‐Density Lipoprotein Receptor‐Deficient Mice by Inhibiting Endothelial VCAM‐1 and ICAM‐1 Signaling

    doi: 10.1161/JAHA.116.004820

    Figure Lengend Snippet: Nox and XO inhibitors and antibodies against VCAM ‐1 and ICAM ‐1 recapitulate the effect of bilirubin on endothelial ROS generation and monocyte transmigration. ROS production by TNF ‐α‐stimulated HUVEC monolayers was assessed by monitoring dihydrorhodamine fluorescence following activation of VCAM ‐1 (α VCAM ‐1) or ICAM ‐1 (α ICAM ‐1), as described in Figure  5 . A and B, Time‐dependent changes in fluorescence intensity following VCAM ‐1 (A) or ICAM ‐1 (B) activation (squares), in the absence (black symbols) or presence of 10 μmol/L of ML 171 (white symbols), or 40 μmol/L of allopurinol ( AP ; gray symbols). Curves reflect mean fluorescence intensity (±SEM) expressed relative to maximal activation at 60 minutes (n=3 sets of experiments). C, Compares the effect of the DMSO vehicle (Veh), 40 μmol/L of AP , and/or 10 μmol/L of ML 171 on THP ‐1 cell migration across HUVEC monolayers, as described in Figure  2 (n=4 sets of experiments). D and E, Results of analogous studies examining THP ‐1 migration in the presence or absence of antibodies against ICAM ‐1 ( ICAM ; 10 μg/mL), VCAM ‐1 ( VCAM ; 10 μg/mL), β 2 (5 μg/mL), and/or α 4 (20 μg/mL). F, Lineweaver–Burk plot of H 2 O 2 produced by isolated XO in the presence of 50 μmol/L of bilirubin ( BR ; diamonds; K i =3.4 μmol/L), 30 μmol/L of the competitive inhibitor, AP (triangles; K i =6.7 μmol/L), 30 μmol/L of the noncompetitive inhibitor, 2‐chloro‐6(methylamino) purine ( CMAP ; squares; K i =4.7 μmol/L), or the DMSO vehicle (circles). Data reflect the mean (±SEM) of 3 sets of experiments. * P

    Article Snippet: Mouse anti‐human VCAM‐1 (clone P3C4) and mouse anti‐human ICAM‐1 (clone P2A4) were purchased from Millipore (Temecula, CA).

    Techniques: Transmigration Assay, Fluorescence, Activation Assay, Migration, Produced, Isolation

    Proposed mechanism of bilirubin modulation of VCAM ‐1‐ and ICAM ‐1‐dependent monocyte migration. Ligation of VCAM ‐1 and ICAM ‐1 with their corresponding integrins, α 4 β 1 /α 4 β 7 and α L β 2 , leads to Rac‐1‐ and calcium (Ca 2+ )‐dependent activation of NADPH oxidase (Nox) and xanthine oxidase ( XO ). These enzymes generate the reactive oxygen species ( ROS ), superoxide (O 2 ˙ − ) and hydrogen peroxide (H 2 O 2 ), that comprise a signaling cascade, which leads to activation of matrix metalloproteinases ( MMP )‐2 and ‐9 and disruption of endothelial tight junctions. Bilirubin, a potent antioxidant that undergoes intracellular redox cycling (dashed lines) through action of biliverdin reductase ( BVR ), scavenges Nox‐ and XO ‐derived ROS , thereby inhibiting leukocyte migration. ICAM‐1 indicates intercellular adhesion molecule 1; Rac1, Ras‐related C3 botulinum toxin substrate 1; VCAM‐1, vascular cell adhesion molecule 1.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Bilirubin Prevents Atherosclerotic Lesion Formation in Low‐Density Lipoprotein Receptor‐Deficient Mice by Inhibiting Endothelial VCAM‐1 and ICAM‐1 Signaling

    doi: 10.1161/JAHA.116.004820

    Figure Lengend Snippet: Proposed mechanism of bilirubin modulation of VCAM ‐1‐ and ICAM ‐1‐dependent monocyte migration. Ligation of VCAM ‐1 and ICAM ‐1 with their corresponding integrins, α 4 β 1 /α 4 β 7 and α L β 2 , leads to Rac‐1‐ and calcium (Ca 2+ )‐dependent activation of NADPH oxidase (Nox) and xanthine oxidase ( XO ). These enzymes generate the reactive oxygen species ( ROS ), superoxide (O 2 ˙ − ) and hydrogen peroxide (H 2 O 2 ), that comprise a signaling cascade, which leads to activation of matrix metalloproteinases ( MMP )‐2 and ‐9 and disruption of endothelial tight junctions. Bilirubin, a potent antioxidant that undergoes intracellular redox cycling (dashed lines) through action of biliverdin reductase ( BVR ), scavenges Nox‐ and XO ‐derived ROS , thereby inhibiting leukocyte migration. ICAM‐1 indicates intercellular adhesion molecule 1; Rac1, Ras‐related C3 botulinum toxin substrate 1; VCAM‐1, vascular cell adhesion molecule 1.

    Article Snippet: Mouse anti‐human VCAM‐1 (clone P3C4) and mouse anti‐human ICAM‐1 (clone P2A4) were purchased from Millipore (Temecula, CA).

    Techniques: Migration, Ligation, Activation Assay, Derivative Assay

    Time course for  TNF ‐α‐induced expression of adhesion molecules by  HUVECs .  HUVEC  monolayers were incubated in the presence of 5 ng/mL of  TNF ‐α (TNF; squares) or the  TNF  vehicle (Veh; circles), and expression of  VCAM ‐1,  ICAM ‐1, E‐Selectin, P‐Selectin, and  PECAM ‐1 was determined at the indicated time points by  qRT ‐ PCR  and western blotting. A, Time‐dependent changes in  mRNA  for E‐Selectin (black symbols) and P‐Selectin (white symbols), while (B) displays the results obtained for  VCAM ‐1 (gray symbols),  ICAM ‐1 (black symbols), and  PECAM ‐1 (white symbols). Data reflect  mRNA  levels (±SEM) relative to untreated cells (n=4 separate sets of experiments). C, Representative immunoblots for E‐Selectin,  ICAM ‐1, and  VCAM ‐1, with graphs (D) quantifying expression at the indicated time points relative to unstimulated cells at time 0 (Con) and corrected for  GAPDH  (n=3 sets of experiments). * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Bilirubin Prevents Atherosclerotic Lesion Formation in Low‐Density Lipoprotein Receptor‐Deficient Mice by Inhibiting Endothelial VCAM‐1 and ICAM‐1 Signaling

    doi: 10.1161/JAHA.116.004820

    Figure Lengend Snippet: Time course for TNF ‐α‐induced expression of adhesion molecules by HUVECs . HUVEC monolayers were incubated in the presence of 5 ng/mL of TNF ‐α (TNF; squares) or the TNF vehicle (Veh; circles), and expression of VCAM ‐1, ICAM ‐1, E‐Selectin, P‐Selectin, and PECAM ‐1 was determined at the indicated time points by qRT ‐ PCR and western blotting. A, Time‐dependent changes in mRNA for E‐Selectin (black symbols) and P‐Selectin (white symbols), while (B) displays the results obtained for VCAM ‐1 (gray symbols), ICAM ‐1 (black symbols), and PECAM ‐1 (white symbols). Data reflect mRNA levels (±SEM) relative to untreated cells (n=4 separate sets of experiments). C, Representative immunoblots for E‐Selectin, ICAM ‐1, and VCAM ‐1, with graphs (D) quantifying expression at the indicated time points relative to unstimulated cells at time 0 (Con) and corrected for GAPDH (n=3 sets of experiments). * P

    Article Snippet: Mouse anti‐human VCAM‐1 (clone P3C4) and mouse anti‐human ICAM‐1 (clone P2A4) were purchased from Millipore (Temecula, CA).

    Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot